Recent in vivo studies have shown that treatments that decrease circulating prolactin (PRL) in rodents result in significant immunosuppression. Our attempts to demonstrate corresponding direct stimulatory effects of PRL on cultured lymphocytes were unsuccessful. However, antibodies against pituitary PRL potently inhibited both murine and human lymphocyte proliferation in response to both T and B cell mitogens. Further studies using IL 2 and IL 4 responsive cell lines (CTLL‐2 and HT‐2) demonstrated that the same anti‐PRL antibodies inhibited the proliferative response to these cytokine growth factors. Thus, antibodies to PRL appear to block an event occurring in the G1 to GS phase transition of these cell lines, which constitutively express growth factor receptors. The inhibitory activity of anti‐PRL antibodies could be adsorbed by addition of purified human PRL or by immobilized PRL on an affinity column. Antibodies to other pituitary hormones were without inhibitory effect on CTLL‐2 cell proliferation. Proliferation of lymphocytes in serum‐free medium was also potently inhibited by anti‐PRL antibodies, suggesting that antibody effects were not due to neutralization of PRL or other factors contained in culture serum supplements. We suggest from these data that a protein with homology to PRL and recognized by these anti‐PRL antibodies is produced by lymphocytes and plays a critical role in their progression through the cell cycle.—Hartmann, D. P.; Holaday, J. W.; Bernton, E. W. Inhibition of lymphocyte proliferation by antibodies to prolactin. FASEB J, 3: 2194‐2202; 1989.