Malonyl-CoA decarboxylase, a normally mitochondrial enzyme, accumulates in the cytoplasm of specialized glands to cause production of multiple methyl-branched fatty acids. Evidence was presented that a single copy of the decarboxylase gene present in the goose genome codes for both the mitochondrial form found in extremely low amounts in the liver and the cytosolic form found in large amounts in uropygial glands. To elucidate how a single gene encodes both forms, the malonyl-CoA decarboxylase gene and the cDNAs for both the mitochondrial (liver) and the cytoplasmic (gland) species were cloned and sequenced. The decarboxylase gene, found in a 21-kb segment of cloned genomic DNA, is composed of five exons of 0.521, 0.118, 0.156, 0.145, and 1.93 kb interrupted by 6.9, 1.5, 0.45, and 9.3-kb introns. Exon 1 revealed two ATGs in frame 150 bp apart. cDNA for the cytoplasmic form and mitochondrial form showed identical nucleotide sequence, except that the latter was longer than the former. The longest cDNA for the cytoplasmic form of the enzyme extended only 44 bp 5' to the second ATG and the position corresponded to the transcription initiation site of the cytoplasmic form revealed by primer extension and RNase protection. The cDNA for the mitochondrial form isolated from the library extended 19 bp further upstream. Primer extension and RNase protection indicated that transcripts for the mitochondrial form initiated upstream from the first ATG. The N-terminal segment of the open reading frame initiated at the first ATG showed an amphipathic signal sequence appropriate for mitochondrial import. A putative full length mRNA for the mitochondrial form of the enzyme when translated in vitro yielded a 55-kDa primary translation product which was processed by removal of about 5 kDa during uptake into goose liver mitochondria. These results strongly suggest that in most tissues transcription initiates 5'- to the first ATG, generating a transcript that would generate a protein with an N-terminal leader for transport into mitochondria. In the uropygial gland the use of an alternate promoter generates transcripts initiated between the two ATGs and the translation product accumulates in the cytoplasm since it lacks a mitochondrial targeting sequence.