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The reference bioassay for lactogens is the Nb2 cell proliferation assay, whose extreme sensitivity allows the detection of very low amounts of lactogenic activity in biologic fluids. The use of rat Nb2 cells raises the problem of species specificity when analyzing lactogens of other origin, including human lactogenic hormones for which no reference bioassay currently exists. In this article, we describe two new homologous bioassays for human lactogens. One is a transcriptional bioassay generated by stably transfecting 293 human embryonic kidney fibroblasts using two plasmids, encoding the human prolactin receptor (hPRLR) and the PRL-responsive lactogenic hormone response element luciferase reporter gene. The second is a proliferation assay obtained by stably transfecting Ba/F3 cells with a plasmid encoding the hPRLR. We provide characterization of the various clones or cell populations that were isolated, and we describe experiments that were performed to achieve optimized protocols for both bioassays. These new assays were compared with other cells types exhibiting well-recognized PRL-mediated responses (proliferation of Nb2 or of human breast tumor cell lines), using various lactogen analogs. This comparative analysis provides strong evidence that the intrinsic characteristics of each bioassay dramatically affect the biologic properties attributed to the lactogen of interest. Depending on the assay, a given analog can exhibit agonistic or antagonistic properties. We hypothesize that in addition to species specificity, assay sensitivity is the key parameter in directing the apparent bioactivity of lactogens. Of course, in the end, it will be necessary to confirm the agonistic or antagonistic properties of the tested analogs, in vivo.