The mouse dihydrolipoamide dehydrogenase (Dld) gene has been cloned, characterized, and mapped. This nuclear gene encodes a mitochondrial protein that is shared among several alpha-keto acid dehydrogenase complexes and the glycine cleavage system. The Dld gene is contained within an approximately 21-kb region and consists of 14 exons ranging in size from 69 to 521 nucleotides. The open reading frame codes for a preprotein of 509 amino acids with a predicted mature protein of 474 amino acids that is highly conserved among mammalian species (> 90% identical). Primer extension analyses have shown the gene to have transcription initiation sites with tissue-specific differences in relative utilization. The 5' flanking region is G-C rich and lacks a TATA box, but does contain initiator element and multiple transcription factor-binding consensus sequences. Northern blot analysis shows that the Dld mRNA in various tissues is approximately 2.4 kb in size. The Dld gene has been localized to the proximal region of chromosome 12, approximately 21 cM from the centromere.