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The Nef gene product is a regulatory protein of HIV whose biological function is poorly understood. Nef has been thought to have a negative effect on viral replication in vitro but has been shown in studies with SIV to be necessary in the establishment of viraemia in vivo. In vitro studies in various human cell lines have shown that Nef downregulates the expression of cell surface CD4 and thus could have effects on the immune response. We have generated four transgenic mouse lines, with constructs containing two different Nef alleles under the control of CD2 regulatory elements to examine the interaction of Nef with the host immune system in vivo. In adult transgenic mice we have found marked downregulation in the level of CD4 on the surface of double positive thymocytes and a decrease in the number of CD4+ T cells in the thymus. Functional analyses have revealed a decrease in the total activation of transgenic thymocytes by anti-CD3 epsilon antibody. By specific intracellular staining of T cells in such mice we have found CD4 colocalizing with a Golgi-specific marker. These results strongly suggest a Nef mediated effect on developing CD4 thymocytes resulting from interference of Nef in the intracellular trafficking or post-translational modification of CD4.