Hydroxysteroid (alcohol) sulfotransferase STa catalyzes the 3-phosphoadenosine 5-phosphosulfate-dependent O-sulfonation of a diverse array of alcohols including neutral hydroxysteroids. Many of the secondary alcohols that interact with this sulfotransferase are the metabolic products of stereoselective oxidation or reduction reactions. The role that the stereochemistry of secondary alcohol substrates plays in the catalytic efficiency of STa was investigated with a series of chiral benzylic alcohols and the enantiomeric 3-hydroxyl-containing steroids, androsterone and epiandrosterone. In the case of (R)-(+)- and (S)-()-enantiomers of 2-methyl-1-phenyl-1-propanol and 1-phenyl-1-butanol, the effect of stereochemistry on the catalytic efficiency of STa was small (less than 2-fold in favor of (R)-(+)-enantiomers). However, as the number of carbons in the -alkyl chain increased, the stereoselectivity for the sulfation of enantiomers increased as well. The (R)-(+)-enantiomers of 1-phenyl-1-pentanol, 1-phenyl-1-hexanol, and 1-phenyl-1-heptanol were preferred as substrates over the (S)-()-enantiomers with a 3-fold difference in catalytic efficiency. STa showed absolute stereospecificity in the sulfation of the enantiomers of 1-phenyl-1-cyclohexylmethanol; (R)-(+)-1-phenyl-1-cyclohexylmethanol was a substrate for STa, while the (S)-()-enantiomer was a competitive inhibitor of the enzyme. Although a lower degree of stereoselectivity was observed with the 3-hydroxyl-containing steroids, androsterone and epiandrosterone, results with these substrates were also consistent with the conclusion that the stereochemistry of secondary alcohols is an important factor in the catalytic efficiency of STa.