A defect in the E1 beta subunit of the branched chain alpha-keto acid dehydrogenase (BCKDH) complex is one cause of maple syrup urine disease (MSUD). In an attempt to elucidate the molecular basis of MSUD, we isolated and characterized the cDNA of the E1 beta subunit of BCKDH. Using the cDNA as a probe, a chromosomal gene related to E1 beta subunit of human BCKDH was isolated from human gene libraries. The gene of E1 beta subunit is over 100 kilobases long and is split into 10 exons. All of the splice donor and acceptor sites conform to the GT/AG rule. The transcription initiation site was determined by nuclease S1 mapping and primer extension and was located 47 bases upstream from the initiation codon. A "CAAT" box and its reverse complement sequences were present at 39 bases and 75 bases upstream from the cap site, but there was no "TATA" box-like sequence. There were three sets of sequences resembling the transcription factor Sp1-binding sites and two sets of sequences resembling the enhancer core sequence. We also analyzed the chromosomal localization of the gene for the E1 beta subunit of BCKDH. The gene was mapped to chromosome 6. Knowledge of the gene structure of human BCKDH E1 beta subunit will facilitate further studies on the expression and regulation of this gene and provide necessary information for analyses of mutations in patients with MSUD.