During the course of isolating, characterizing, and cloning estrogen and 3-hydroxysteroid sulfotransferases from the guinea pig adrenal gland, it was noted that cytosolic preparations from this tissue would also sulfonate testosterone. Therefore, we set out to isolate and clone the enzyme that performs this reaction. Testosterone sulfotransferase (TST) was isolated from the guinea pig adrenal by using the standard procedures of ion exchange, affinity, and high-performance liquid chromatography. When purified, TST was examined by liquid-phase nondenaturing isoelectric focusing, it was found that the TST activity profile completely overlapped with the activity profile of the 3alpha-hydroxysteroid sulfotransferase (3alphaHST) isoform, but not the 3beta-hydroxysteroid sulfotransferase (3betaHST) isoform. This finding was further investigated by overexpressing the cDNAs for 3alphaHST and 3betaHST in Escherichia coli and examining the expressed proteins for TST activity. This experiment confirmed that 3alphaHST does indeed function as a TST. In addition, 3alphaHST was also found to sulfonate estradiol but not estrone, a finding that further suggested that 3alphaHST may function as a general 17beta-hydroxysteroid sulfotransferase.