Gel filtration and anion-exchange chromatography have been used to investigate whether 3'-phosphoadenylylsulfate:bile salt sulfotransferase activity from female rat and hamster liver is heterogeneous. Using these techniques at least three different enzyme activities were demonstrated with two different bile salt substrates. In both animals, but particularly the rat, there was a marked difference in the substrate specificity between each of the peaks of enzyme activity. The reducing agent, 2-mercaptoethanol, enhanced the proportion of the highest molecular weight (130 000) form of the enzyme from rat liver detected with glycochenodeoxycholate as substrate. This effect was duplicated by alkylation of sulfhydryl groups with iodoacetamide and is interpreted as being due to intermolecular association caused by disruption of intramolecular disulfide bonds.