The in vitro metabolism of purified (RP-1 standard) and secreted rat prolactin (PRL) was compared using the pigeon crop-sac bioassay and a radioimmunoassay (RIA). The secreted hormone was obtained by incubating rat adenohypophyses in medium 199. The two forms of PRL were incubated in medium 199 without serum or with 20% rat serum. Explants of rat kidney, liver or lactating mammary gland were added to some of the flasks containing the PRL in the different media. In general, the secreted PRL was found to be more readily immunoinactivated than was the purified hormone and incubation with tissues increased the degree of inactivation. Addition of serum or bovine serum albumin to the medium reduced the degree of RIA inactivation. The biological activity of the RP-1 was reduced by incubation with mammary tissue in one experiment. However, with secreted hormone, incubation with tissues resulted in an increase in biological activity. These results indicate that secreted rat PRL is immunologically less stable than the purified pituitary form of the hormone. Furthermore, the loss of immunoactivity of the secreted PRL may be accompanied by 'activation' of its biological activity.