The peptide hormone, prolactin, when added to organ explants of rat mammary gland, rapidly (within 1 h) induced the accumulation of casein mRNA. Casein mRNA sequences, as determined by hybridization with a specific cDNA probe, were shown to increase for up to 48 h after prolactin addition. The magnitude of this response was dependent upon the day of pregnancy at which the tissue was placed in culture. Maximal levels of induction (as great as 45-fold) were obtained using tissue from 15-day pregnant rats. Further data indicate that two steroid hormones, hydrocortisone and progesterone, were able to modulate the prolactin-induced accumulation of casein mRNA. The continuous presence of hydrocortisone was not necessary for prolactin induction of casein mRNA. However, the presence of hydrocortisone was required for maximal accumulation of casein mRNA. The induction of casein mRNA by prolactin was inhibited in a dose-dependent manner by the simultaneous addition of progesterone to the organ culture. Thus, hydrocortisone appears to potentiate the prolactin induction of casein mRNA, whereas progesterone is able to prevent casein mRNA accumulation. Since mammary gland organ culture is performed in a serum-free, chemically defined medium, this system allows a detailed examination of the mechanims by which a peptide hormone regulates the rapid accumulation of a specific mRNA.