In-situ hybridization and Northern blot hybridization were used to identify mRNA for pituitary prolactin in mammary tissue obtained from female rats 1 day before expected parturition, 1 day after parturition and on day 7 of lactation. Prolactin cDNA was labelled with 32P for Northern analysis and with digoxigenin for in-situ hybridization. Total and poly(A)+ RNA from pituitary, mammary and control (fat and kidney) tissues were analysed by agarose gel electrophoresis with transfer to nitrocellulose and hybridization to a cDNA for rat prolactin. Although present in much smaller amounts than the 1.0 kb transcript in pituitary RNA homogenates, mammary RNA homogenates from all three stages contained mRNA of approximately 1.0 kb which hybridized with the prolactin probe. Similar analyses of fat and kidney failed to reveal any hybridization at the 1.0 kb size. When tissue sections were hybridized to the cDNA probe, specific hybridization was observed in the milk secretory cells of the mammary alveoli and the lactotroph cells of the anterior pituitary, but not in liver cells or in RNAase-treated sections of mammary tissue. In summary, these results demonstrate that milk secretory cells of the rat mammary gland transcribe the gene for prolactin, and they raise the possibility that a primary target tissue for blood-borne prolactin may also synthesize prolactin.