Assay conditions for measurement of human skin fibroblast branched-chain L-amino acid aminotransferase activity were established and applied to studies on subcellular distribution and kinetic properties of the enzyme. Digitonin fractionation of cultured cells revealed that the aminotransferase activity was mainly (at least about 95%) associated with mitochondrial citrate synthase activity. As tested with L-leucine, activity of the enzyme against amino group acceptors (forward reaction) was in the order 2-oxoglutarate [Symbol: see text] branched-chain > straight-chain 2-oxo acids (C3-C8). With 4-methyl-2-oxopentanoate, activity against amino group donors (reverse reaction) was in the order L-glutamate [Symbol: see text] branched-chain > straight-chain (C2-C6) and other L-amino acids. The data suggest that, in human fibroblasts, isoenzyme type I resides within the mitochondrial space. Possible implications for the metabolism of branched-chain compounds are discussed.