A 4175-bp EcoRI fragment of DNA that encodes the a and / 3 chains of the pyruvate dehydrogenase (lipoamide) component (El) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the Ela subunit (368 amino acids, M , 41 312, without the initiating methionine residue) and E l p subunit (324 amino acids, M , 35 306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1p gene begins just 3 bp downstream from the Ela stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The Ela and E l p subunits of the B. stearotherrnophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the Elm and EIP subunits of pyruvate and branched-chain 2-0x0-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the El a chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.The 2-0x0-acid dehydrogenase multienzyme complexes catalyse the oxidative decarboxylation of pyruvate, 2-0x0-glutarate and branched-chain 2-0x0 acids, releasing C 0 2 and generating the corresponding acyl-CoA and NADH. The three constituent enzymes of the pyruvate dehydrogenase (PDH) complex are pyruvate dehydrogenase (lipoamide) (El), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). In the PDH complexes from Gram-positive bacteria and mitochondria, the E l components are composed of E l a and E l p polypeptide chains and the E2 components form cores consisting of 60 copies of the E2 polypeptide chains arranged with icosahedral symmetry. These cores bind multiple copies of the E l and E3 components tightly but non-covalently (for recent reviews see [I] and [2]).The E l component catalyses the decarboxylation of pyruvate and the reductive transfer of the acetyl group to a lipoic acid moiety which is covalently bound to the E2