Medium-chain acyl-CoA dehydrogenase (MCAD) is one of the three straight-chain length-specific dehydrogenases involved in the first step of fatty acid oxidation. Inherited defects of acyl-CoA dehydrogenases occur in humans, and MCAD deficiency is the most common. We have cloned the coding and 3' untranslated sequence of mouse MCAD cDNA. The mouse MCAD cDNA coding region is 1263 bp long with a 3' untranslated region of 576 bp and encodes a 421 amino acid precursor protein. Comparing the nucleotide and deduced amino acid sequences of the mouse MCAD cDNA to rat and human MCAD cDNAs reveals considerable similarity between species. Amino acid residues where substitutions result in human MCAD deficiency are conserved in the mouse. Amino acid residues involved in important enzymatic functions are also conserved.