In this study, we describe the cloning and characterization of a soluble form of kynurenine aminotransferase (KAT, EC 126.96.36.199) present in rat brain. Soluble KAT was purified from rat kidney and the amino acid sequences of four tryptic peptides determined. These peptides were found to belong to the amino acid sequence reported for rat kidney soluble cysteine conjugate beta-lyase, indicating that rat kidney KAT and beta-lyase represent the same molecular entity. Oligonucleotide probes derived from the beta-lyase cDNA were then used as primers for PCR of reverse-transcribed rat brain poly(A)+ RNA. After subcloning of the resulting PCR fragment and sequencing of the isolated rat brain clone, its oligonucleotide sequence was found to be identical to that reported for the beta-lyase cDNA. Further evidence that the isolated rat brain clone encoded for KAT was obtained by transfecting HEK-293 cells with a construct containing the coding sequence for the enzyme. The transfected cells exhibited KAT activity and, in the presence of 2 mM pyruvate and 2-oxoglutarate, the Km values for L-kynurenine were 1.2 mM and 86.3 microM, respectively. Northern blot analysis of rat kidney, liver, and brain RNA revealed a single species of KAT/beta-lyase mRNA of approximately 2.1 kb.