We have investigated the effect of infusion of DL-beta-hydroxybutyrate (BOHB) (30 mumol X kg-1 X min-1) on glucose and free fatty acid (FFA) metabolism by means of the primed constant infusion of [U-14C]glucose and [1,2-13C]palmitic acid. The role of the hormonal response to the ketone infusion was assessed by controlling the hormone levels pharmacologically. In one group hormones were not controlled, while in the other two groups insulin and glucagon were maintained at constant levels by infusion of somatostatin, insulin, and glucagon at constant rates. In one of these hormonally controlled groups, combined alpha- and beta-adrenergic blockade was also employed. BOHB infusion increased total ketone concentration approximately 10-fold and, when hormones were not controlled, induced a significant increase in glucagon concentration. Regardless of hormonal status, elevation of the ketone levels decreased the rate of glucose production and FFA appearance. Glucose oxidation decreased in proportion to the reduction in the rate of glucose uptake in all groups. When sympathetic activity was not blocked an increase in the percent of FFA uptake oxidized enabled the percent CO2 production from FFA oxidation to remain constant despite the decrease in FFA uptake. However, when sympathetic activity was blocked the increase in the percent of FFA uptake oxidized observed in the other groups was prevented. We conclude from these studies that an elevation in ketone levels directly affects glucose and FFA metabolism independent of changes in insulin and glucagon levels and sympathetic activity.