Serum gamma-glutamyl transferase (GGT) was separated into nine to 11 isoenzyme bands (designated as GGT I-XI) by vertical slab electrophoresis on polyacrylamide gradient gel. The diagnostic value of GGT isoenzyme II (GGT II) for hepatocellular carcinoma (HCC) was studied, and the results were as follows: 1) GGT II was positive in 90% of 90 cases of HCC, and negative in most patients with acute and chronic viral hepatitis, extrahepatic tumors, in pregnant women, and in healthy controls; 2) the positive rate of GGT II assay was higher than that of alkaline phosphatase isoenzyme I (ALP I), alpha-fetoprotein (AFP), and alpha 1-antitrypsin (AAT) in 101 cases of HCC. In cases in which the AFP was greater than 50 ng/ml or less than 50 ng/ml, the positive rates of GGT II were 70.8% and 75-100%, respectively; 3) of 14 cases of small-size HCC, the positive rate of GGT II was 78.6%, which was higher than that of AFP (50%), AAT (28.6%), and ALP I (0%); 4) of 62 cases that were false-positive for GGT II assay, 24.2% developed into HCC during a follow-up of 2.1-20 months. In subjects with persistent and recurrent positivity of GGT II, 86.7% and 22.2%, respectively, developed HCC. No patient with temporal positivity of GGT II developed HCC. The results show that GGT II can be applied as an additional marker for HCC, and is valuable not only for the diagnosis of clinical HCC, but for the detection of small or subclinical HCC. Periodic follow-up with assay of GGT II in patients at high risk for HCC may predict the development of hepatoma.