The production of milk is closely linked to nutritional state in many mammalian species, but the mechanisms by which changes in nutritional state are signaled to the mammary glands are poorly understood. Simultaneous measurements of mammary blood flow and glucose arterio-venous difference were made across the inguinal mammary glands of anesthetized, lactating rats. Blood flow to the mammary glands of previously fed rats was 0.48 mL/min per gram of mammary tissue. Glucose supply was 1.7 mumol/min per gram and 28% was extracted by the mammary glands. After food deprivation for 18 h, mammary blood flow decreased 48%, glucose arterio-venous difference decreased 72%, and hematocrit increased 7%, resulting in a 60% decrease in glucose supply and an 88% decrease in glucose uptake. After 1 h of refeeding, glucose supply had returned to a similar level to that of normally fed animals, but glucose uptake was 60% higher than in the normally fed state. Mammary glucose uptake was not closely linked to either blood flow or glucose supply, suggesting that substrate supply was not the primary determinant of mammary metabolism. Denervation experiments showed that the mammary metabolic response to altered nutritional state was also unlikely to be closely controlled by neural pathways. Severance of the cutaneous branch of the posterior division of the femoral nerve innervating the inguinal mammary glands did not reduce the high glucose uptake by mammary glands of either fed or refed rats, nor did denervation change the low glucose uptake by mammary glands of food-deprived rats. Denervation reduced blood flow in the associated mammary gland, however, indicating that neural pathways may play a role in supporting mammary blood flow when food is available. In in vitro experiments, the rate of glucose uptake was 35% lower in mammary acini from food-deprived rats than in fed rats 2.5 h after tissue removal, indicating some persistence of the food deprivation-induced suppression of mammary metabolism. Administration of insulin increased glucose uptake in acini from both fed and food-deprived rats, indicating that insulin may be involved in signaling the mammary gland of the restoration of nutrient supply when food-deprived rats are refed. The effects of administration of a gut extract in vivo and in vitro are discussed.