The conversion of L-threonine to glycine in both prokaryotes and eukaryotes takes place through a two-step biochemical pathway involving the enzymes L-threonine dehydrogenase (EC 1.1.1103) and 2-amino-3-ketobutyrate coenzyme A ligase (KBL; EC 2.3.1.29). The genes encoding these enzymes have been described in prokaryotes but not in eukaryotes. We report the cloning of transcripts for KBL, the second enzyme in the pathway, from human and murine lung and a partial transcript from bovine liver. Two peptide sequences from the purified bovine KBL protein, one from the N-terminus and the other from the peptide containing the pyridoxal 5'-phosphate-binding lysine residue [Tong, H. & Davis, L. (1994) J. Biol. Chem. 269, 4057-4064], are identical with regions of the conceptual translation of the transcript obtained from bovine liver. The partial transcript from bovine liver was very similar to the human transcript, being 91% and 92% identical at the nucleotide and amino-acid levels, respectively. The human and murine KBL transcripts are 1.5 kb long, with ORFs encoding proteins of 419 and 416 residues, respectively. The mouse protein has 90% identity with the human protein. The human transcript is strongly expressed in heart, brain, liver and pancreas compared with the lung. The N-termini of both human and mouse proteins have characteristics of mitochondrial import sequences. Both human and murine proteins have 54% identity with the well-characterised prokaryote KLB protein from Escherichia coli. Database searches with the human cDNA sequence enabled us to identify the human KBL gene on chromosome 22q12-13, consisting of nine exons over 9 kb, and a hypothetical Caenorhabditis elegans KLB gene on chromosome IV, consisting of five exons over 2 kb.