Cholesterol can undergo sulfate conjugation to form cholesterol 3-sulfate. Our experiments were performed to determine whether human liver cytosol could catalyze the sulfation of cholesterol, and, if so, whether any of the three well-characterized human hepatic cytosolic sulfotransferases, dehydroepiandrosterone sulfotransferase (DHEA ST), thermostable (TS) phenol sulfotransferase (PST), or thermolabile (TL) PST might participate in the reaction. On the basis of substrate kinetics, two "forms" of cholesterol sulfotransferase (CST) activity were present in human liver cytosol, one with high and one with low affinity for cholesterol. Apparent KM values of the high- and low-affinity activities were 0.14 and 15 microM for cholesterol and 0.30 and 0.19 microM for 3'-phosphoadenosine-5'-phosphosulfate, respectively. Both kinetic forms of CST activity had thermal inactivation profiles similar to those of DHEA ST and TS PST, but both were more thermostable than was TL PST. Enzyme inhibition studies performed with 2,6-dichloro-4-nitrophenol (DCNP) showed that inhibition profiles for both high- and low-affinity CST activities were similar to those of DHEA ST and TL PST, but both were more resistant to DCNP inhibition than was TS PST. Experiments performed with 20 individual human liver samples confirmed these observations and demonstrated highly significant correlations between both high- and low-affinity CST activities and DHEA ST activity (rs = 0.740, p = 0.0001 and rs = 0.767, p < 0.0001, respectively). However, the level of activity of neither kinetic form of CST activity was significantly correlated with either TS or TL PST activities.(ABSTRACT TRUNCATED AT 250 WORDS)