We searched expressed sequence tag databases with conserved domains of the short-chain alcohol dehydrogenase superfamily and identified another isoform of 17β-hydroxysteroid dehydrogenase, 17βHSDXI. This enzyme converts 5α-androstane-3α, 17β-diol to androsterone. The substrate has been implicated in supporting gestation and modulating γ-aminobutyric acid receptor activity, 17βHSDXI is colinear with human retinal short-chain dehydrogenase/reductase retSDR2, a protein with no known biological activity (accession no. AAF06939). Of the proteins with known function, 17βHSDXI is most closely related to the retinol-metabolizing enzyme retSDR1, with which it has 30% identity. There is a polymorphic stretch of 15 adenosines in the 5′ untranslated region of the cDNA sequence and a silent polymorphism at C719T. A 17βHSDXI construct with a stretch of 20 adenosines was found to produce significantly more enzyme activity than constructs containing 15 or less adenosines (43% vs. 26%, P <0.005). The C719T polymorphism is present in 15% of genomic DNA samples. Northern blot analysis showed high levels of 17βHSDXI expression in the pancreas, kidney, liver, lung, adrenal, ovary, and heart. Immunohistochemical staining for 17βHSDXI is strong in steroidogenic cells such as syncytiotrophoblasts, sebaceous gland, Leydig cells, and granulosa cells of the dominant follicle and corpus luteum. In the adrenal 17βHSDXI, staining colocalized with the distribution of 17α-hydroxylase but was stronger in the mid to outer cortex. 17βHSDXI was also found in the fetus and increased after birth. Liver parenchymal cells and epithelium of the endometrium and small intestine also stained. Regulation studies in mouse Y1 cells showed that cAMP down-regulates 17βHSDXI enzymatic activity (40% vs. 32%, P < 0.05) and reduces gene expression to undetectable levels. All-trans-retinoic acid did not affect 17βHSDXI expression or activity, but addition of the retinoid together with cAMP significantly decreased activity over cAMP alone (32% vs. 23%, P < 0.05). Cloning and sequencing of the 17βHSDXI promoter identified the potential nuclear receptor steroidogenic factor-1 half-site TCCAAGG-CCGG, and a cluster of three other potential steroidogenic factor-1 half-sites were found in the distal part of intron 1. Collectively, these results suggest a role for 17βHSDXI in androgen metabolism during steroidogenesis and a possible role in nonsteroidogenic tissues including paracrine modulation of 5α-androstane-3α, 17β-diol levels. 17βHSDXI could act by metabolizing compounds that stimulate steroid synthesis and/or by generating metabolites that inhibit it.