Using 1-14C-labeled substrates, the metabolism of naturally occurring branched-chain L-amino acids was studied in incubations with cultured human skin fibroblasts derived from normal subjects and from a patient with maple syrup urine disease (variant form). Practically saturating conditions were reached at 1 mmol/liter of substrate and metabolic rates remained essentially constant up to 120 min. In control fibroblasts, the transamination of 14C-labeled leucine, valine, isoleucine, and allo-isoleucine (1 mmol/liter) was about 26, 13, 12, and 5 nmol/90 min/mg of cell protein, respectively. The portion of transamination products undergoing oxidative decarboxylation within the cells was about 17, 43, 34, and 23%, respectively. With the maple syrup urine disease cell line, comparable transamination rates were found. As compared to the findings with normal cells, however, 14CO2 production from the above mentioned substrates was reduced and amounted to 14, 11, 25, and 45%, respectively. Thus it appeared that residual branched-chain 2-oxo acid dehydrogenase activity was differently reduced towards the four 2-oxo acid substrates.