Most enzyme deficiencies in humans are inherited as autosomal recessive traits. The term dominant negative is applied to mutant alleles in which a mutant protein interferes in one way or another with the function of the normal protein being produced from the wild-type allele in a heterozygote. Such a dominant negative effect usually involves homomeric or heteromeric proteins. 3-Methylcrotonyl-CoA carboxylase (MCC) is a heteromeric mitochondrial enzyme comprised of biotin containing MCCalpha subunits and smaller MCCbeta subunits, encoded by the genes MCCA and MCCB, respectively. Mutations in these genes cause isolated MCC deficiency, an autosomal recessive disorder with a variable phenotype ranging from severe neonatal to asymptomatic adult forms. Patients with MCC deficiency have a characteristic organic aciduria with greatly increased excretion of 3-hydroxyisovaleric acid (3-HIVA) and 3-methylcrotonyl-glycine (3-MCG). Here, two patients with elevated excretion of 3-MCG and 3-HIVA and partial deficiency of MCC are discussed, one of them with severe neurological symptoms. Both showed evidence of biotin responsiveness and were heterozygous for the missense mutation MCCA-R385S. Evidence is presented that MCCA-R385S is a dominant negative allele leading to biochemical abnormalities and clinical symptoms in heterozygous individuals and that it is responsive to pharmacological doses of biotin in vivo.