The effect of dietary arginine deficiency on rat mammary gland development was investigated in pregnant rats and ovariectomized nonpregnant rats. Consumption of an arginine-deficient diet from day 0 of pregnancy significantly reduced the total DNA and RNA content of the mammary gland by day 20. Pair-feeding studies revealed that reduced food intake associated with consumption of the arginine-deficient diet was not responsible for the altered nucleic acid content of the gland. Restriction of food intake by pair-feeding did reduce the wet weight of the mammary gland as well as its fat content when compared with ad libitum fed controls, but the degree of reduction did not fully account for the depressing effect on the wet weight and fat content observed when the arginine-deficient diet was fed. The ratio of RNA to DNA in the mammary tissues of rats fed the arginine-deficient diet indicated normal protein synthetic activity per cell. The extent of reduction in mammary nucleic acid content by arginine deficiency was similar for pregnant rats and ovariectomized nonpregnant rats treated with estrogen and progesterone. These studies show that dietary arginine is required for optimal mammary growth. The possible mechanism(s) by which arginine can modify mammary growth are discussed.