A possible autocrine function of prolactin (Prl) in human breast cancer was explored by the addition of a panel of anti-human Prl mAbs to T47Dco and MCF7 human breast adenocarcinoma cells. mAb 631 and mAb 390 inhibited cell growth by 86 and 68%, respectively, in the estrogen receptor-negative T47Dco cells and by 20 and 71%, respectively, in the estrogen receptor-positive MCF7 cells. Conditioned medium prepared from T47Dco cells was assessed for the presence of Prl-like molecules by its ability to stimulate growth of Prl-responsive Nb2 rat lymphoma cells. Growth of Nb2 cells under the influence of human Prl or conditioned medium was abolished when either solution was pretreated with mAb 390, followed by Immunobead precipitation (Bio-Rad, Melville, NY). T47Dco cells secrete 0.7 microgram lactogen/ml over a 24-48-h period. With the use of reverse transcription-PCR, an expected 612-bp band was detected by ethidium bromide staining, and its similarity to pituitary Prl was confirmed by Southern blot analysis with the use of human Prl cDNA as a probe. A single M(r) 22,000 band, the dominant size of monomeric pituitary Prl, was found in immunoprecipitates of both cell extracts and conditioned medium from T47Dco cells labeled metabolically with [35S]cysteine. These data suggest that human breast cancer cells synthesize and secrete biologically active Prl.