Human proline oxidase (HsPOX), a mitochondrial inner membrane oxidase, catalyzes the first step of the proline degradation pathway. Deficiency of HsPOX causes type I hyperprolinemia (HPI), an autosomal recessive disorder characterized by accumulation of proline. To understand the biochemistry of HsPOX and the molecular basis of HPI, we cloned human POX by homology probing of the EST database with sequences conserved in D. melanogaster and S. cerevisiae POXs (DmPOX and ScPOX, respectively) and using the relevant EST clones to screen human cDNA libraries. Surprisingly, we identified two different forms of putative full length human POX cDNAs (HsPOX1 and HsPOX2, respectively). HsPOX1 has an 1608-base pair open reading frame encoding a protein of 536 residues with a predicted molecular mass of 59 kDa. HsPOX2 has an 1800-base pair open reading frame encoding a protein of 600 residues with a predicted molecular mass of 66 kDa. The deduced amino acids of HsPOX1 has 36.3% and 27.3%, and of HsPOX2 48.5% and 21.6% sequence identity with those of DmPOX and ScPOX, respectively. On Northern blots of RNA from multiple human tissues, HsPOX1 detects a 1.8 kb transcript only in liver and kidney; HsPOX2 detects a 2.4 kb transcript in nearly all tissues, including brain and kidney but not in liver. We mapped HsPOx1 to 19q13.1 and HsPOX2 to 22q11.2. Functional expression, and mutation analysis of HPI patients are in progress.