Biochemical studies in five patients with a defect in biotin-responsive holocarboxylase synthesis are reported. The age of onset (2 d to 6 y) as well as the severity of illness varied considerably. In all patients diagnosis was established by the finding of organic aciduria typical for multiple carboxylase deficiency in a catabolic state. In four patients the response to biotin therapy was evaluated by measurement of mitochondrial carboxylase activities in lymphocytes and by monitoring urinary organic acid excretion. In three patients clinical symptoms disappeared with 10-20 mg biotin/d, whereas normalization of the biochemical parameters required higher doses (20-40 mg/d). The fourth patient required a dose of 100 mg biotin/d before her skin rash disappeared. She remains mentally retarded and shows slightly elevated urinary organic acid excretion. Carboxylase activities were clearly deficient in fibroblasts grown in the commonly used medium which contains 10 nmol/L biotin (contributed by FCS in medium) in two patients. Fibroblasts of the other three patients became deficient only in a low biotin medium (0.1 nmol/L). Reactivation of deficient carboxylase activities in relation to time and biotin concentration correlated well with the severity and age of onset of illness in four patients. In one patient, however, carboxylase reactivation followed a more complex pattern requiring the longest incubation time but only a moderately increased biotin concentration of 19 nmol/L compared with 3-5 nmol/L in normal cells and 34-4000 nmol/L in the other four patients. The results in the five patients are in accordance with a primary defect of holocarboxylase synthetase due to a decreased affinity for biotin, in one patient combined with a decreased Vmax.