Mammary tissue from midpregnant mice will undergo cytodifferentiation in vitro in completely defined medium when stimulated with insulin, hydrocortisone and prolactin (IHP). After a preliminary incubation without hormones (17–21 hr) the initial effects of these hormones on macromolecular synthesis were investigated. Addition of IHP enhances the incorporation of radioactive uridine, amino acids and galactose into trichloroacetic acidinsoluble material within 1 hr; thymidine incorporation is enhanced several hours later. By establishing a pool of radioactive uridine before the addition of the hormones, it was shown that at least part of the enhanced rate of uridine incorporation into acid-insoluble material was due to increased synthesis and not changes in the soluble pools. Of the 3 hormones, insulin alone produces the same augmentation of uridine and thymidine incorporation as IHP during the first 10 hr after the hormonal additions. The fact that the rise in thymidine incorporation is delayed is used along with other data to suggest that insulin may play a permissive role in this system with regard to DNA synthesis. (Endocrinology85: 747, 1969)