Previous work has shown that insulin is necessary for lactation. In this study, the short term effects of insulin on the net metabolism of glucose and amino acids by the bovine mammary gland were examined in six experiments in lactating cows. To prevent insulin-induced hypoglycemia from masking the direct effects of insulin, glucose (50--75 g/h) was infused with insulin 12 U/h). The insulin plus glucose infusion (2 h) was followed by a 2-h infusion of glucose alone (75 g/h). Throughout the control and infusion periods, arterial and mammary venous blood samples were obtained to measure venoarterial concentration differences of glucose and amino acids across the mammary gland. During the insulin plus glucose infusion, insulin concentrations increased from basal values of 9 +/- 1 to 38 +/- 6 mu U/ml (mean +/- SE). During glucose infusion alone, the insulin concentration was 15 +/- 2 mu U/ml. Glucagon concentrations were not significantly altered by the treatments. The extraction of glucose by the mammary gland was not altered significantly by insulin, nor was the extraction altered during the infusion of glucose alone. During the infusion of insulin plus glucose and glucose alone, the extraction ratios [(A -- V)/A] of isoleucine, leucine, tyrosine, aspartate, and valine were increased 10--20%. Thus, it appears that insulin does not influence glucose extraction and only moderately may enhance the extraction of some amino acids by the bovine mammary gland.