The hormonal requirements for lactogenesis were investigated using explant cultures of mammary tissue obtained from cows at 30-40 days prepartum. Hormones used were insulin, hydrocortisone, and prolactin, and parameters examined were radioactive acetate incorporation into fatty acids, secretory response ratings, and histological and ultrastructural analysis. Data indicated that insulin was essential for mammary epithelial cell survival, but insulin alone did not result in the intiation of milk synthesis. The culture of explants in a medium containing insulin plus hydrocortisone resulted in alterations in the cytology of alveolar cells but no induction of milk synthesis. Biosynthesis results and secretory response ratings indicated that the initiation of milk synthesis occurred when explants were cultured in insulin and prolactin; however, synthesis was limited and alveolar integrity was not well maintained. Results from all parameters demonstrated that the maximal lactogenic response was obtained when the culture medium contained insulin, hydrocortisone and prolactin. After 48 h of culture in this medium, the rate of acetate incorporation into fatty acids had increased 3-fold and the markedly distended alveolar lumina contained many fat droplets and abundant eosinophilic staining secretion. However, an unusal amount of casein-like micelles and especially lipid also accumulated in the alveolar cells. The accumulation of milk components within the epithelial cells may be related indirectly to the accumulation of products in the lumina or perhaps related to a difference in the hormonal requirement between the initiation of milk synthesis and initiation of milk secretion.