Alanine-glyoxylate aminotransferase (AGT) 2 is a pyridoxal 5'-phosphate dependent, mitochondrial enzyme which, in the rat, is expressed at a high level in the kidney. The amino acid sequences of nine tryptic and seven CNBr peptides of the rat kidney AGT2 were determined. Three overlapping cDNAs encoding the AGT2 were cloned on the basis of its partial amino acid sequences by means of a polymerase chain reaction-based approach involving rat kidney poly(A)+ RNA. The complete cDNA sequence comprised 1,919 bases, and contained a 1,536-base open reading frame which encodes a polypeptide of 512 amino acid residues with a putative presequence consisting of 39 amino acid residues at the amino terminus, giving a precursor protein with a molecular mass of 57,150 Da. The sequence of AGT2 exhibits significant homology with neither peroxisomal AGT1 from human liver nor mitochondrial AGT1 from rat liver. However, the sequence of AGT2 exhibited 30.8, 29.2, and 27.1% identity with those of Escherichia coli 4-aminobutyrate aminotransferase, rat ornithine aminotransferase, and Pseudomonas cepacia 2,2-dialkylglycine decarboxylase, respectively. The active site sequences were also well conserved among these aminotransferases. AGT2, thus, is more similar to the other aminotransferases than to AGT1. The results suggest that the rat kidney AGT2 may play a biological role in amino acid metabolism distinct from that of AGT1.