An in vitro system has been developed to examine the effects of PRL on the normal primate mammary gland. alpha-Lactalbumin a milk protein, was found in breast tissue samples from 17 of 19 primates representing several Macaca and Papio species; concentrations ranged from 10-768 ng/mg protein. That none of the animals was pregnant or lactating and half were nulliparous indicates that milk protein production takes place under normal circumstances, even in breast tissue of nonlactating animals. Studies of the effect of PRL on alpha-lactalbumin production in these tissues in organ culture revealed that PRL maintained existing or stimulated new production of alpha-lactalbumin for periods of up to 9 days. Measurement of alpha-lactalbumin in medium bathing mammary tissue from three animals revealed that mean alpha-lactalbumin production during days 7-9 when PRL was added (100 and 1000 ng/ml) was 11 and 59 times greater, respectively, than control. Simultaneous measurement of tissue concentrations of alpha-lactalbumin revealed that those tissues maintained with PRL (1000 ng/ml) had a mean concentration of alpha-lactalbumin that was 61 times that of controls without PRL. PRL consistently maintained or increased alpha-lactalbumin production in tissues from all 22 primates tested. Even in those premenarchal animals in whose mammary tissue alpha-lactalbumin was undetectable initially, PRL stimulated alpha-lactalbumin production in a dose-related fashion. In contrast, when PRL was absent from medium, alpha-lactalbumin concentrations decreased at 9 days to less than 20% of the initial 3-day value in all cases. These studies provide evidence that mammary tissue from normal nonlactating, nonpregnant primates produces milk proteins and that when tissues are exposed to PRL in culture, production of alpha-lactalbumin is stimulated.