Protein synthesis in the rat mammary gland has been studied using acini isolated from mammary tissue by collagenase digestion. When the acini were incubated with radioactively labeled amino acids, both cellular and milk proteins were synthesized and milk proteins were secreted into the incubation medium. Antisera to the lipogenic enzyme, fatty acid synthase, and the milk proteins, alpha-lactalbumin and the caseins, raised in rabbits, were shown to be specific by analyzing immunoprecipitates on sodium dodecyl sulfate--polyacrylamide gels. The rates of synthesis and secretion of each protein by acini prepared from rats during late gestation and at specific stages of lactation reflect their previously observed concentration in the mammary gland or milk of rats at the corresponding stage of gestation or lactation. Rats were treated according to one of the following regimes between d 7 and 14 of lactation: they were fed a control (20% casein) or a low protein (10% casein) diet ad libitum, they were fed the control diet restricted to 25 g/d (40% of the voluntary intake), they were fed the control diet for 5 d and starved for 48 h or they were treated as in 3 and then refed the control diet ad libitum for 24 h. Food restriction and starvation both resulted in lowered rates of synthesis of all proteins examined compared with either the control or refed animals. Starvation also lowered the rates of secretion of the milk proteins. Consumption of the low protein diet caused a specific decrease in both the rates of synthesis and secretion of alpha-lactalbumin compared with the control rats without affecting the synthesis and secretion of the caseins.