We have analyzed the expression of prolactin receptors (PRL-R) on murine lymphoid cells by using flow cytofluorometry analysis with biotinylated anti-PRL-R mAb raised against several epitopes of the extracellular domain of the PRL-R and by using polymerase chain reaction amplification. We demonstrated that PRL-R were universally expressed in normal rat and mouse hematopoietic tissues. In both primary lymphoid organs, namely, thymus and bone marrow, > 90% of cells were labeled by the anti-PRL-R mAb, but the density of PRL-R (assessed by fluorescence intensity) was lower on thymocytes than on bone marrow cells. In peripheral lymphoid organs there were smaller proportions of cells bearing PRL-R and we could clearly distinguish cell subsets of various fluorescence intensities. By using classical markers for lymphoid cell populations, we noted that all B cells and macrophages from spleen, lymph nodes, and blood strongly expressed the PRL-R. Regarding T cell populations, large proportions (> 85%) of PRL-R+ cells were detected in the four thymocyte subsets, thus contrasting with the smaller proportions (50 to 65%) of T cells labeled in the periphery. Similar percentages of PRL-R+ cells were observed in CD4+ and CD8+ peripheral lymphocyte subsets. Importantly, the stimulation of thymocytes and spleen cells with the T cell mitogen Con A promoted an enhancement of the density of PRL-R molecules on the cell membranes. Because hyperprolactinemia is associated with some autoimmune diseases, we also investigated PRL-R expression in the NZB autoimmune mouse. In contrast to the pattern observed in normal animals, the frequencies of PRL-R-bearing T cells as well as the density of PRL-R per cell increased with age in NZB mice, suggesting that some imbalances of PRL/PRL-R interaction might occur in autoimmune situations. In conclusion, our data provide a molecular basis for a better understanding on the mode of action of PRL within the immune system in physiologic and pathologic situations.