3'-Phosphoadenosine 5'-phosphosulphate: galactosylceramide sulfotransferase which catalyzes the sulfation of galactosylceramide (GalCer) was partially purified from a rat kidney light membrane fraction, and the properties were studied with special reference to substrate specificity. In order to study minimum molecular requirement of the acceptor substrate for the sulfotransferase, analogues of galactosylceramide where omega-amiocaproic or omega-aminododecanoic acid is substituted for the acyl moiety of the native glycolipid were chemically synthesized by improved procedures. The artificial glycolipids were sulfated effectively by the kidney enzyme, suggesting that the synthetic compounds will serve for affinity ligands for the purification of the enzyme. The extent of sulfation in the synthetic compounds was comparable with that of galactosylceramide containing normal acids and higher than that of galactosylsphingonine in which one of hydrocarbon chains is deleted from the native glycolipid. Through substrate specificity experiments, the sulfotransferase has relatively broad specificity acting on beta-linked galactosides at nonreducing ends of mono- and disaccharides which bind, at least, one hydrocarbon chain. Enzyme kinetic analysis by competition assay using mixed acceptors demonstrated that the same, single sulfotransferase catalyzes sulfation of galactosylceramide, lactosylceramide and galactosylsphingosine. As to human cancer, the sulfotransferase activity was hardly detectable in Wilms' tumor tissues that contrasts with renal cell carcinoma tissues where the markedly elevated level was previously demonstrated. When sera from patients with various cancers were examined for the enzyme level, many cases of hepatocellular carcinoma showed significantly increased activity, whereas the hepatoma tissues had hardly detectable level of the enzyme. These observations suggest that a humoral factor derived from the hepatoma induces the sulfotransferase.