Lactose synthetase (LS) is a complex of alpha-lactalbumin and N-acetylglucosamine galactosyl transferase (GT). LS is the rate-limiting enzyme in lactose biosynthesis. Lactose is the main osmotic component of milk and may thereby affect milk volume. Dietary protein inadequacy reduces milk volume and lactose content. The study investigates the role of LS activity in these phenomena. Lactating rats were fed adequate and inadequate amounts of protein of high (milk protein) and low (cereal protein) quality. After 14 days LS and GT activity of mammary tissue was measured. Milk volume and lactose content was determined. While GT activity was unaffected by diet, LS activity was reduced by low dietary protein quality. Addition of bovine alpha-lactalbumin to tissue incubates largely restored the reduced LS activities. Milk volume was affected in a parallel manner to LS activity. A highly significant positive correlation existed between LS activity and total lactose production (r = 0.794; p less than 0.001). Dietary protein inadequacy indirectly reduces milk volume by means of a direct effect on the lactalbumin part of the LS complex and, subsequently, lactose biosynthesis.