Mammalian morphine 6-dehydrogenase (M6DH)1 converts morphine into a reactive electrophile, morphinone. M6DH belongs to the aldo-keto reductase (AKR) superfamily, but its endogenous substrates and entire amino acid sequence remain unknown. A recent rabbit genomic sequencing predicts three genes for novel AKRs (1C26, 1C27 and 1C28) that share >87 amino acid sequence identity and are similar to the partial sequence of rabbit liver M6DH. We isolated cDNAs for the three AKRs, and compared the properties of their recombinant enzymes. Like M6DH, only AKR1C26 that shares the highest sequence identity with hepatic M6DH oxidized morphine. The three AKRs showed NAD +-dependent dehydrogenase activity towards other non-steroidal alicyclic alcohols and 3a/17?-hydroxy-C18/C 19/C21-steroids, and their mRNAs were ubiquitously expressed in rabbit tissues. The kinetic constants for the substrates suggest that at least AKR1C26 and AKR1C28 act as NAD+-dependent 3a/17?-hydroxysteroid dehydrogenases. AKR1C27 differed from AKR1C28 in its high Km values for the substrates and low sensitivity towards competitive inhibitors (ikarisoside A, hinokitiol, hexestrol and zearalenone), despite their 95 sequence identity. The site-directed mutagenesis of Tyr118 and Phe310 in AKR1C27 to the corresponding residues (Phe and Ile, respectively) in AKR1C28 produced an enzyme that was similar to AKR1C28, suggesting their key roles in ligand binding.