We have reported the isolation of genomic clones encoding serine:pyruvate aminotransferase (SPT; also named alanine:glyoxylate aminotransferase, AGT) (T. Oda, T. Funai, and A. Ichiyama, 1990, J. Biol. Chem. 265: 7513-7519). These clones contained the entire SPT/AGT gene of 10 kb. In this work, we characterized this gene. The SPT/AGT gene consists of 11 exons, and the exon-intron boundaries have typical splice donor and acceptor sequences. Determination of the nucleotide sequence up to -1.25 kb from the transcription initiation site revealed the presence of many putative cis elements, some of which may explain the transcriptional regulation of the SPT/AGT gene by glucagon and glucocorticoid. The nucleotide sequence around the 5' flanking region of the rat SPT/AGT gene and the whole gene organization were compared with those of the human SPT/AGT gene. No obvious similarities were observed in the 5' flanking region up to -1.25 kb from the initiation site of the gene, but exons 2 to 10 of the rat and human genes have identical sizes and show high similarities.