To investigate the contribution of branched-chain amino acids as a nitrogen source for alanine in vivo, dogs were infused with L-[15N]leucine, L-[U-14C]leucine, L-[2,3,3,3-2H4]alanine, and D-[6,6-2H2]-glucose, 14C and 15N isotopic equilibrium in plasma leucine, and deuterium enrichment in arterial and femoral plasma glucose and alanine were achieved within 3 h of initiation of the respective isotope infusion in all animals. The average flux of leucine determined by [15N]-leucine was 5.4 μmol.kg-1.min-1, whereas using [14C]-leucine it was 3.7 μmol.kg-1.min-1. Turnover rates for alanine and glucose were 11.0 and 17.2 μmol.kg-1 min-1, respectively. [15N]alanine was detected as early as 30 min, but nitrogen isotopic equilibrium in alanine was not achieved until 6 h. The absolute rate of leucine nitrogen transfer to alanine was 1.92 μmol.kg-1.min-1, which represented 41-73% (mean 53%) of leucine's nitrogen and 15-20% (mean 18%) of alanine nitrogen. Fractional extraction of alanine and leucine by the dog hindlimb was 35 and 24%, respectively. Average net alanine balance was -6.7 μmol.leg-1.min-1, reflecting a release rate (17.4 μmol.kg-1.min-1) that exceeded the rate of uptake (10.8 μmol.leg-1.min-1). Of the leucine taken up by the hindlimb, 34% transferred its nitrogen to alanine and 8% was oxidized to CO2. Since tha latter value reflects transamination as well as irreversible catabolism, the nitrogen derived from the oxidation of leucine by the hindlimb could account for only 25% of the observed 15N incorporation into alanine. The significantly faster flux of leucine nitrogen when compared with leucine carbon suggests significant recycling of the leucine α-ketoacid. These studies demonstrate that leucine is a major donor of nitrogen to circulating alanine in vivo.