In many mammalian cells, efficient introduction of genetic material can be accomplished with recombinant retroviral vectors. Retrovirus vectors have also been used as vehicles for gene therapy studies, since many therapeutic applications require integration of genes into cellular DNA (15, 35). Important potential targets for gene modification are hematopoietic stem cells that have the ability to establish long-lived and multilineage reconstitution of hematopoiesis in mammals (23, 40). Despite advantages of both retroviral vectors and hematopoietic stem cells as tools for genetic therapy, retroviral gene transfer into primitive stem cells of large animals via retrovirus vectors has been problematic, and the potential therapeutic use of gene transfer technology in human diseases remains largely unfulfilled (30). Retrovirus-mediated gene transfer into human hematopoietic stem cells is influenced by multiple factors, including low viral titer (12, 25, 44) and the cycle status of the target stem cells (36). Recently, increasing attention has been focused on the level of expression of viral receptors in these primitive cells. Two different retroviral receptors have been extensively used for targeting cells of the human hematopoietic system: the amphotropic murine leukemia virus (MLV) receptor and the gibbon ape leukemia virus (GALV) receptor (31). Both receptors have been characterized as sodium-dependent phosphate symporters (5, 45). Significant sequence similarity exists between the amphotropic receptor and the GALV receptor, although these receptors bind virion particles without cross-interference (37). The level of expression of the amphotropic receptor, Pit-2, has been measured by mRNA levels and appears to be low in human and primate CD34+ hematopoietic cells (21, 43, 51) and in murine hematopoietic stem cells (42). This has led to speculation that a low level of expression of the receptor protein is responsible, at least in part, for low-level transduction with amphotropic-packaged vectors (21, 42, 49). In contrast, the mRNA level of the GALV receptor, Pit-1, appears to be higher than that of the amphotropic receptor in CD34+ cells isolated from nonhuman primates (21). Thus the levels of mRNA of these two receptors appear to correlate with the respective levels of infection in different cells by recombinant retroviral vectors (21, 49). Kiem et al. (22) have exploited this difference to improve transduction of primate hematopoietic stem cells with GALV-pseudotyped vectors. Previous studies have demonstrated that the capacity of retroviruses to bind their cognate receptor is sensitive to a variety of manipulations. During infection with replication-competent retrovirus, receptor occupancy by envelope protein interferes with subsequent reinfection of cells, a process termed superinfection interference (48). Transduction with replication-defective viruses, which lack envelope protein coding sequences, has rarely been reported to be associated with receptor interference (28). However, receptor interference may be particularly important with respect to the functional consequences of low receptor density in hematopoietic cells, a major target population for gene therapy applications. Since viral occupancy of the cognate receptor is a critical component of efficient infection, our laboratory has exploited colocalization of viral particles and target cells via interaction with fragments of the extracellular matrix protein fibronectin (FN) to increase transduction of a variety of mammalian cells, including hematopoietic cells (17, 39, 46). In a recent study, we noted that continuous exposure of human hematopoietic cells to virus in the presence of the peptide fragment FN CH-296 prior to induction of cell cycling led to a transduction efficiency significantly inferior to that when the cells were stimulated with cytokines prior to virus exposure (16). Since current clinical protocols frequently utilize multiple infections targeting the same receptor, the possibility that receptor expression or receptor interference may play a role in inefficient transduction of hematopoietic cells, particularly in the presence of fibronectin, could be critical to gene therapy protocols. In the study reported here, hematopoietic cell lines and primary human CD34+ hematopoietic cells were infected on FN CH-296 simultaneously with distinguishable retroviral vectors pseudotyped with either the same or different envelope proteins. Simultaneous infection with two viruses pseudotyped with the same envelope protein was associated with inhibition of transduction both in human erythroleukemia (HEL) cells and in human CD34+ primary hematopoietic cells. This inhibition was attributable to receptor interference, since no inhibition could be demonstrated during simultaneous infection by two vectors utilizing different receptors for cell entry. Receptor interference continued for up to 24 h after the initial exposure to virus. In contrast, simultaneous infection with MLV amphotropic and GALV-pseudotyped vectors yielded transduction efficiencies consistently higher than in infection with either vector alone in both target cells. These studies demonstrate that, in the presence of FN CH-296, functional receptor density limits transduction of hematopoietic cells and sequential infection of these cells within a short period of time provides little additional quantitative transduction benefit. Finally, simultaneous infection with vectors targeting different receptors may prove to be an additional approach for further improving transduction of hematopoietic targets for gene therapy protocols.