Fetal calf serum (FCS) enhances the synthesis and secretion of prolactin (PRL) by explants of human decidua incubated in Gey's balanced salt solution. In order to further characterize the factors involved in the prolactin-stimulating activity (PSA) of serum, decidua was incubated in Gey's buffer alone and in buffer containing either FCS, bovine serum albumin (BSA), or human serum (HS). When buffer was supplemented with varying concentrations of FCS, ad dose-related PSA was apparent between 0.1% and 10%. The PSA of FCS was not altered by eithr heating (85degrees C for 30 minutes), boiling, or dialysis. The protein fraction of FCS, precipitated with trichloroacetic acid, redissolved in dilute NaOH, and dialyzed against Dulbecco's phosphate buffer, demonstrated PSA comparable to that of whole serum. A similar concentration of BSA added to Gey's buffer did not stimulate the production of PRL by decidua. The PSA of human serum obtained from euprolactinemic men and women was not different from that of FCS. These data indicate that FCS and HS contain a substance(s) (1) which stimulates the secretion of PRL by human decidua, (2) which is very stable, being resistant to dialysis, boiling, and denaturation by trichloroacetic acid, (3) and which is most likely a large polypeptide with a molecular weight greater than 12,000 or a smaller moiety tightly bound to a protein.