To evaluate the prognostic significance and clinicopathologic correlation of proliferative activity in patients with hepatocellular carcinoma, Ki-67 antigen expression was examined using immunohistochemical staining with monoclonal antibody M1B1. Seventy-two patients (65 men, 7 women; age range 24–77 years, mean, 52 years) having hepatocellular carcinoma surgically resected were studied. Tumor and nontumorous tissues were stained with monoclonal antibody MIB1 with microwave oven pretreatment. Tumor and nontumor MIB1 (T-MIB1 and NT-MIB1) scores were assessed by counting the positive staining nuclei per 1,000 cells. The T-MIB1 score ranged from 5–630 per 1,000 cells (mean ± standard deviation [SD] = 145 ± 162). It was found to be significantly higher in less well-differentiated tumors (Edmondson’s grades III and IV) than in well- differentiated ones (Edmondson’s grades I and II) (P = .017). The T-MIB1 score was also higher in nonencapsulated tumors than in encapsulated ones, although it did not reach statistical significance (P = .069). It had no influence on tumor size, tumor invasiveness, the background disease in the nontumorous livers, patients’ HBsAg status, or serum a-fetoprotein levels. Diseases in the nontumorous livers or patients’ HBsAg status had no influence on the NT-MIB1 scores. When the tumors were stratified into two groups with T-MIB1 score ≤, 20 and T-MIB1 score > 20, those patients with score ≤ 20 had significantly longer disease-free survival (DFS) than those with scores > 20 (median DFS: 34 months and 4.7 months, respectively; P = .011). In addition, MIB1 and PCNA were closely correlated (P < .01). The authors conclude that proliferative activity in hepatocellular carcinoma, as defined by MIB1 immunohistochemical analysis, is significantly related to tumor cellular differentiation. It is also a potentially valuable prognostic factor in patients with this tumor.