Previous studies have shown that target tissues cleave rat prolactin (rPRL) to form a two-chain derivative that yields approximately 16- and approximately 6-kDa fragments upon reduction. Both cleaved rPRL and the purified 16-kDa fragment have novel biological activities. Thus, cleavage may be of physiological significance. We determined whether normal mammary tissue or stroma (lacking mammary epithelial cells [MEC]) or mammary tumor tissue could cleave rPRL in vitro. Tumor explants were derived from a transplantable mammary tumor cell line (TPDTMT-4EP). The hormone was incubated with explants of those tissues from female BALB/c or DDD mice. Medium samples were processed by SDS-PAGE, and the relative abundance of intact and cleaved rPRL was determined by densitometric analysis of immunoblots using an antiserum to the 16-kDa fragment of rPRL. After 2-hr of incubation, normal DDD mammary explants cleaved approximately 25% of added rPRL, but tumor explants cleaved none. Explants of intact virgin BALB/c mouse mammary gland and of parenchyma-free "cleared" mammary fat pad cleaved rPRL equally well, but explants of abdominal adipose tissue had low cleaving activity. Homogenates of cleared mammary fat pads that were incubated with rPRL contained a high proportion of the cleaved form but none of the free 16-kDa fragment. These results indicate that cleavage of rPRL is highly specific to mammary stroma. Such cleavage may yield a form of the hormone that has novel activities within the mammary gland or elsewhere in the body.