Pyruvate carboxylase (PC) is a biotinylated mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate. Children with inborn errors of PC metabolism have lactic acidosis, hypoglycemia, and mental retardation. The variable severity of the clinical phenotype is dependent on both genetic and environmental factors. Specific mutations causing human PC deficiency have not been published. Two consanguineous families with milder forms of PC deficiency were characterized at the biochemical and molecular levels. In both families, the probands were found to have low PC activity(range: 7-20% of control) associated with normal protein and mRNA levels. In the first case, the proband apppeared to improve after administration of biotin. Sequencing of patient-specific PC cDNA demonstrated a T to C substitution at nucleotide 434, which causes a valine to alanine change at amino acid residue 145 [sequence based on Wexler et al. Biochim. Biophys. Acta 1227: 46-52, 1994]. Direct sequencing of the parents showed that they were heterozygous for this mutation. In the second case, a brother and sister had mental retardation and episodes of severe lactic/ketoacidosis. In this family, a C to T substitution at nucleotide 1351 results in a cysteine for arginine substitution at amino acid residue 451. In both families, no other mutations were found and both substitutions occurred in conserved amino acid residues. These mutations, located in protein domains involving ATP and pyruvate binding, provide a unique opportunity to analyze how natural occurring mutations effect PC function.