A new isoform of rat liver estrogen sulfotransferase (EST), rEST-6, which is distinct from the previously reported rat EST [Demyan et al., Molec. Endocrinol. 6 (1992) 589], has been cloned, expressed, purified and characterized. A PCR procedure using oligonucleotide primers synthesized to the 5'-nontranslated and 3'-nontranslated regions of the published rEST sequence was used to isolate rEST-6 cDNA. The cloned DNA is 1000 bp in length and encodes a protein of 295 amino acids with a calculated molecular mass of 35,300 Da. rEST-6 is selectively expressed in male rats, as confirmed by Northern blot and immunoblot analyses. Northern blot analysis of male and female rat liver RNA with the rEST-6 cDNA as a probe shows a band with male RNA but not with female RNA. Similarly, immunoblot analysis of male and female rat liver cytosols with an antibody to rat EST yields a strong immunoreactive band in rat liver cytosol from male rats but not from females. Subsequent to bacterial expression and purification of rEST-6, the enzyme was analyzed kinetically and shown to sulfate estrogens but not dehydroepiandrosterone, pregnenolone, cortisol or testosterone. Maximal sulfation activity towards both beta-estradiol and estrone occurred at a concentration of 1 microM with substrate inhibition at higher concentrations. These results indicate that multiple, closely related forms of EST are present in rat liver. Analysis of the activity and regulation of these different EST enzymes is important in understanding estrogen metabolism in rats.