Holocarboxylase synthetase is one of two enzymes known to be involved in the metabolism of biotin. It catalyses the fixation of biotin to inactive apocarboxylases yielding active carboxylases. Deficiency of this enzyme leads to multiple carboxylase deficiency which is fatal in the absence of prompt diagnosis and treatment with biotin. In a pregnancy at risk for deficiency of holocarboxylase synthetase prenatal diagnosis was performed by assay of the enzyme in amniocytes. The K m for biotin was 62โข8 nM which was 12 times the control value of 5โข0 nM. The V max was 2 per cent of the control value. This was confirmed by assay of the activity of propionyl CoA carboxylase (20-26 per cent control), 3-methylcrotonyl CoA carboxylase (14-19 per cent control) and pyruvate carboxylase (12-30 per cent control) and demonstration of biotin responsiveness in vitro. All carboxylase activities were restored to 51-58 per cent of control when amniocytes were cultured in medium containing 1 M biotin. Diagnosis was ultimately confirmed by assay of holocarboxylase synthetase in lymphocytes from the infant after birth. The K m for biotin of the holocarboxylase synthetase of the infant was 60โข3 nM while that of a parallel control was 6โข9 nM. Prenatal treatment of the mother with biotin led to a concentration of biotin of 240 nM in the serum of the infant at birth that was four times the K m of the enzyme for biotin. The infant was clinically well at birth, and organic acid analysis of the blood and urine revealed no accumulation of the characteristic metabolites.