AB - Steroid glucuronidation was investigated in solubilized female rat and rabbit liver microsomes and in preparations of UDP-glucuronsyltransferase (UDPGT) activity resolved from these organelles. Solubilized rabbit liver microsomes possessed relatively high UDPGT activity towards estrone and β-estradiol but not testosterone. Glucuronidation observed at the 3-OH position of β-estradiol is 20-fold greater than at the 17-OH position. Estrone UDPGT activity, highly purified from rabbit liver microsomes, was active towards estrone and the 3-OH position of β-estradiol but not towards testosterone (17-OH) or the 17-OH position. Thus, estrone UDPGT activity demonstrated essentially the same specificity for conjugation of the 3-OH position of β-estradiol as observed in microsomes. Solubilized liver microsomes from female rats possessed approximately 4-fold more activity towards testosterone (17-OH) than estrone (3-OH). Rat liver microsomes formed about -fold more β-estradiol 17-glucuronide than 3-glucuronide. Following a Chromatofocusing procedure where a gradient from pH 9-7 was employed, an eluant fraction was obtained that was enriched in estrone UDPGT activity relative to testosterone UDPGT activity. The fraction preferentially conjugated the 3-OH position of β-estradiol. Two eluant fractions were obtained which demonstrated high levels of UDPGT activity towards testosterone and β-estradiol but not estrone. Both fractions possessed a UDPGT activity that displayed a high degree of specificity for conjugation of the 17-OH position of β-estradiol. A 17-OH steroid UDPGT was purified to apparent homogeneity from one of these fractions. These results suggest that separate UDPGT activity exists in female rat liver for the glucuronidation of the 3- and 17-OH positions of β-estradiol. In female rabbit liver, β-estradiol is predominantly conjugated at the 3-OH position by a single form of UDPGT.