Mouse kidney contains two 3(17)alpha-hydroxysteroid dehydrogenases (HSDs) that show essentially the same properties except for their isoelectric points. However, the structural differences and physiological roles of the two enzymes remain unknown. In this study, we have isolated cDNAs for the two 3(17)alpha-HSDs from a total RNA sample of mouse kidney by reverse transcription-PCR. The identity of the cDNAs was confirmed by characterization of the recombinant enzymes that showed the same molecular weights, pI values, pH optima, substrate specificity and inhibitor sensitivity as those of the enzymes from mouse kidney. We also found that the recombinant enzymes reduce precursors of neuroactive progesterone derivatives, 5alpha-dihydrotestoserone, deoxycorticosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate and estrone at low Km values of 0.3-2 microM. The two enzymes belonged to the aldo-keto reductase (AKR) family, and their 323-amino acid sequences differed only by five amino acids. The sequences of the two isoforms are identical to those of proteins that are predicted to be encoded in a gene for AKR1C21 in the database of the mouse genome. However, the mRNAs for the two isoforms were expressed in mouse kidney and other tissues, in which their expression levels were different. The results indicate an important role of 3(17)alpha-HSD in controlling the concentrations of various steroid hormones in the mouse tissues, and suggest the existence of two genes for the two isoforms of the enzyme.