Human N-acetylglucosaminyltransferase-III (GnT-III) has not been hitherto purified from either tissue or cell line. Although rat GnT-III has been purified from rat kidney tissue, the procedure involves laborious and complex steps. We have developed a simplified procedure based on a QAE (diethyl[2-hydroxypropyl]aminoethyl)-Sepharose and an immunoaffinity chromatography technique. The immunoaffinity chromatography utilized a monoclonal antibody to human aglycosyl recombinant N-acetylglucosaminyltransferase-III. With the new procedure, human N-acetylglucosaminyltransferase-III was purified from hepatocellular carcinoma tissues with an increase in the specific enzyme activity of 378-fold.