Androgens and estrogens play major roles in cell differentiation, cell growth, and peptide secretion in steroid target tissues. In addition to the binding of these hormones to their receptors, formation and metabolism are important in the action of steroids. Metabolism of the potent steroid hormones includes glucuronidation, a major pathway of steroid elimination in liver and several steroid target tissues. Glucuronidation is catalyzed by UDP-glucuronosyltransferases (UGTs), which transfer the polar moiety from UDP-glucuronic acid to a wide variety of endogenous compounds, including steroid hormones. The UGT superfamily of enzymes is subdivided into two families, UGT1 and UGT2, on the basis of sequence homology. To date, six UGT2B proteins have been isolated, namely UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, and UGT2B17, all of which have been demonstrated to be active on steroid molecules, except for UGT2B10 and UGT2B11, for which no substrate was found. The relative activity of these enzymes on steroidal compounds remains unknown due to variable levels of UGT2B expression in different in vitro cell line models and various conditions of the enzymatic assays. Comparison of the glucuronidation rates of these enzymes requires a unique system for UGT2B protein expression, protein normalization, and enzymatic assays. In this study we have stably expressed UGT2B4, UGT2B7, UGT2B15, and UGT2B17 in the HK293 cell line, which is devoid of steroid UGT activity; characterized their kinetic properties relative to UGT protein expression; determined their transcript and protein stabilities; and established extensively their tissular distributions. UGT2B7 was demonstrated to glucuronidate estrogens, catechol estrogens, and androstane-3alpha,17beta-diol more efficiently than any other human UGTB isoform. UGT2B15 and UGT2B17 showed similar glucuronidation activity for androstane-3alpha,17beta-diol (30% lower than that of UGT2B7), whereas UGT2B17 demonstrated the highest activity for androsterone, testosterone, and dihydrotestosterone. UGT2B4 demonstrates reactivity toward 5alpha-reduced androgens and catechol estrogens, but at a significantly lower level than UGT2B7, 2B15, and 2B17. Cycloheximide treatment of stably transfected HK293 cells demonstrated that the UGT2B17 protein is more labile than the other enzymes; the protein levels decrease after 1 h of treatment, whereas other UGT2B proteins were stable for at least 12 h. Treatment of stable cells with actinomycin D reveals that UGT2B transcripts are stable for 12 h, except for the UGT2B4 transcript, which was decreased by 50% after the 12-h incubation period. Tissue distribution of the UGT2B enzymes demonstrated that UGT2B isoforms are expressed in the liver as well as in several extrahepatic steroid target tissues, namely, kidney, breast, lung, and prostate. This study clearly demonstrates the relative activities and the major substrates of human steroid-metabolizing UGT2B enzymes, which are expressed in a wide variety of steroid target tissues.