Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inborn error of mitochondrial fatty acid oxidation. To determine if immunoreactive enzyme protein is present in patients with MCAD deficiency, we studied cultured skin fibroblasts from patients with the 985 point mutation, present in about 85% of cases, and cell lines from patients in which the point mutation is not present or only involves one allele. Immunoblotting studies, using a polyclonal antibody to the purified protein, showed an absence of immunoreactive protein in mitochondrial fractions prepared from fibroblasts from MCAD-deficient patients. To determine whether MCAD protein accumulated in the cytosol because of impaired transport into the mitochondria, we immunoprecipitated MCAD protein from the fibroblast homogenate. MCAD protein was detected in the immunoprecipitates from controls, but not in those from the MCAD-deficient patients. These results suggest that either the MCAD protein is not synthesised or, if produced, it is rapidly degraded.