Medium-chain acyl-coenzyme A dehydrogenase (MCAD; mouse gene Acadm; human gene ACADM) catalyzes the initial step of fatty acid beta-oxidation in mitochondria. Inherited MCAD deficiency is an autosomal recessive disorder that occurs at high frequency in humans and is associated with considerable morbidity and mortality. We have cloned and characterized mouse Acadm which spans approximately 25 kb and contains 12 exons. The promoter region does not contain TATA or CAAT boxes and is G + C-rich (60%) within 200 bp of the cap site. A CpG island extends from 5' of the transcription start point into intron 1. The 5' regulatory region and a portion of intron 1 contain several Sp1 consensus sites and three regions containing hexamer DNA sequences that match the binding consensus for steroid/thyroid nuclear receptors. These putative nuclear receptor response elements (NRRE) share DNA sequence homology and electrophoretic mobility shift characteristics with known NRRE in the human ACADM promoter [Carter et al., J. Biol. Chem. 268 (1993) 13805-13810]. We have mapped mouse Acadm to the distal end of chromosome 3. Sequences previously localized to chromosome 8 are shown to be a pseudogene, and an additional pseudogene was identified on chromosome 11.