In vitro glucuronidation of 2-hydroxyestriol has been investigated by means of HPLC with dual-electrode coulometric detection. When incubated with rat or dog liver microsomal preparation in the presence of UDPGA, 2-hydroxyestriol was transformed into the 2-glucuronide together with a small amount of 16- and/or 17-glucuronides. In contrast, incubation of 2-hydroxyestriol with guinea-pig liver microsomal preparation yielded the 3-glucuronide and a trace amount of the 2-glucuronide, but no ring D glucuronides. Upon pretreatment with 3-methylcholanthrene male rat liver exhibited a marked increase in both 2- and 3-glucuronidation activities, whereas female rat liver showed an elevation only in 2-glucuronidation. On the other hand, in male and female rats pretreatment with phenobarbital caused a relatively small increase in the glucuronidation activity of the liver. In the male guinea-pig, glucuronidation was not affected by pretreatment with either of the two compounds. The present result demonstrates the multiplicity of hepatic 2-hydroxyestriol UDP-glucuronyl-transferase in the rat, guinea-pig and dog.