To determine directly the interconversion of circulating leucine and alpha-ketoisocaproate (KIC) in vivo, as well as the effects of fasting on leucine and KIC metabolism, 14- and 96-h fasted dogs were studied during simultaneous infusion of L-[4,5-3H]leucine and [U-14C]KIC. The specific radioactivities of 3H- and 14C-labeled leucine and KIC were determined and transfer rates calculated using a two-pool reversible model. In both groups, approximately 32% of the total leucine carbon entering was converted to KIC, whereas 60% of circulating KIC is converted to leucine. Plasma [3H]KIC specific radioactivity was only half of the circulating [3H]leucine specific activity, suggesting entry of KIC from an unlabeled pool. Fasting decreased the leucine-KIC interconversion, entry of unlabeled KIC, and irreversible loss of KIC. These data demonstrate that interconversion of circulating leucine and KIC is extensive and that fasting decreases the conversion of leucine to KIC in the labeled and unlabeled KIC pools, suggesting that transamination is a major regulator of leucine metabolism in fasting dogs.