PRL can induce interleukin-2 (IL-2) receptor expression in splenocytes from ovariectomized (OVX) female rats. In this further study of the effects of PRL on lymphocytes in vitro we found that PRL induced IL-2 receptor expression, IL-2 production, and proliferation of splenocytes and thymocytes from OVX rats. Cells from male rats were not affected. The proliferative response, as measured by [3H]thymidine incorporation, depended on the concentration of PRL and the presence of adherent cells in the culture. After a 48-h incubation with PRL (1 microgram/ml), splenocytes from OVX rats incorporated essentially the same amount of [3H]thymidine as cells incubated with the polyclonal T-cell mitogen Concanavalin-A (ConA). As determined by autoradiography, approximately 40% of the splenocytes responded to PRL or to ConA. After incubation of splenocytes and thymocytes with PRL, bioactive IL-2 was detected in culture medium only from cells of OVX female rats, while incubation with ConA caused IL-2 production by lymphocytes from both male and OVX rats. However, ConA induced IL-2 activity sooner than PRL. Immunofluorescent-flow cytometric analysis revealed time-dependent increases in percentages of IL-2 receptor-positive splenocytes as well as increases in percentages of total T-cells and cells of the CD8 and, to a lesser extent, the CD4 subclass after PRL stimulation.