The high concentrations of dehydroepiandrosterone sulfate and pregnenolone sulfate in the mammalian brain, despite the blood-brain barrier's impermeability to these compounds, and the apparent independence of these concentrations from those in plasma prompted us to investigate whether enzymatic sulfation of dehydroepiandrosterone was detectable in the rat brain. Low hydroxysteroid sulfotransferase activities were detectable in in vitro incubations of homogenates from all rat brain regions except the cerebellum, being highest in the hypothalamus and pons. This activity was not ascribable to enzyme in brain capillary blood. The activity was mainly cytosolic, although there was also significant activity in the partially purified nuclear fraction. The enzyme had different properties from those of hepatic isozymes, with a pH optimum of 6.5 and a high Km of approximately 2 mM for dehydroepiandrosterone. The enzyme was also active with pregnenolone as substrate. Activities in the brain were approximately 300-fold lower than in the liver but, as in the liver, these were higher in females than in males. The variations in brain activity as a function of age did not parallel those in the liver. Relatively high activities were found in the fetal brain and declined at birth, while activities were insignificant in the fetal liver and rose following birth. There was a major peak in activity in pubertal female brains, but this peak was less important, and later, in males. No evidence was found to indicate that the low brain enzyme activities and high Km were attributable either to the presence of an inhibitor or to the steroid sulfation actually being a secondary activity of another brain sulfotransferase. We discuss whether the sulfotransferase activities found are adequate to synthesize the dehydroepiandrosterone and pregnenolone sulfate found in brain.