Late-onset multiple carboxylase deficiency is characterized clinically by skin rash, alopecia, seizures and ataxia and occasionally by candidiasis and developmental delay. Biochemically, these individuals exhibit findings consistent with a combined deficiency of the biotin-dependent carboxylases. We have found that the activity of the enzyme biotinidase is also deficient in the sera of five affected children (0 to 3% of mean control activity, 5.80 ± 0.89 nmol · min⁻ · ml⁻ serum), and believe that it represents the primary biochemical defect in this disease. Biotinidase catalyzes the removal of biotin from the ϵ-amino group of lysine, through which biotin is covalently bound to the four known human carboxylases, thereby regenerating biotin for reutilization. The deficient activity in our patients was not due to an inhibitor, particularly biotin. It is also not a consequence of feedback control in affected individuals under treatment with pharmacologie doses of biotin. The biotinidase activities of the parents of those children who were available for study were intermediate between deficient and normal values (46% to 65% of mean normal activity). Children lacking biotinidase activity are unable to recycle biotin, and are thus entirely dependent upon exogenous biotin to prevent deficiency. Our findings indicate that the primary biochemical defect in late-onset multiple carboxylase deficiency is in biotinidase activity which is inherited as an autosomal recessive trait.