Eight distinct forms of the soluble 17alpha-hydroxy steroid dehydrogenase of rabbit liver were resolved by DEAE-cellulose chromatography and isoelectric focusing. Five of these enzymes were homogeneous as judged by polyacrylamide-gel electrophoresis. Substrate-specificity studies carried out with oestradiol-17alpha and oestradiol-17alpha 3-glucuronide revealed a variation in activity toward these substrates among the different purified enzyme forms. Three forms of the 17alpha-hydroxy steroid dehydrogenase exhibited equal activity toward both oestrogen substrates, whereas three forms of the enzyme displayed a greater activity toward the glucuronide derivative of oestradiol-17alpha. One enzyme in particular is essentially specific for oestradiol-17alpha 3-glucuronide, its activity toward oestradiol-17alpha being only one-thirtieth that observed with the 3-glucuronide derivative.