Initially, it was observed that whereas the propionyl CoA carboxylase (PCC) activity in peripheral blood leukocytes of a 13-month-old girl with isolated PCC deficiency who had been given biotin supplements (4.5 mg/day) for 6 months was only 3% of control, her βMCC activity was greatly elevated at 20 times control activity. After biotin supplements were discontinued, βMCC activity in leukocyte extracts declined over 3 wk to values just above the normal range with no associated change in PCC activity; readministration of biotin caused her βMCC activity to rise in 3 days to the initial elevated values. These results prompted measurement of PCC and βMCC activities in leukocyte extracts of three normal adults given 20 mg biotin orally per day. Their PCC and βMCC activities increased 2− to 3-fold and 4− to 9-fold, respectively, within 7–10 days. After biotin was discontinued, both enzyme activities returned to normal in 4–5 wk. The effect of high concentrations of biotin and βMCC activities was also investigated in cultured fibroblasts from three control subjects. PCC activity increased only 11% in normal fibroblasts grown in medium containing fetal bovine serum and high concentrations of biotin (900 μg/ml). However, PCC and βMCC activities were increased 70–75% and 120–140%, respectively, when confluent normal fibroblasts were incubated in medium containing 10% human serum and 0.1–1.0 μg/ml biotin. This stimulation was independent of the fibroblast growth cycle. Furthermore, cycloheximide failed to inhibit the increase in PCC activity caused by biotin, suggesting that the enhanced activity in fibroblasts was not due tode novocarboxylase synthesis. These studies demonstrate that high concentrations of biotin can stimulate biotin-dependent carboxylases in human tissues, possibly by converting apocarboxylase to holocar-boxylase or by activating preexisting inactive holocarboxylase.SpeculationThe stimulation of human propionyl CoA and β-methylcrotonyl CoA carboxylase activities in peripheral blood leukocytesin vivoand in cultured skin fibroblasts by high concentrations of biotin represents a promising model for investigating the cellular metabolism of biotin in man.