Protein induced vitamin K absence or antagonist II (PIVKA-II) and alpha-fetoprotein (AFP) have been considered useful serum markers of hepatocellular carcinoma (HCC). In this study, we examined the clinicopathologic significance of these tumour markers in patients with HCCs by measuring their serum levels and performing immunohistochemistry. We studied 349 Japanese patients with HCCs. Their serum PIVKA-II and AFP levels were determined by enzyme immunoassay before treatment. We examined the correlations between serum PIVKA-II and AFP levels and tumour size, presence of satellite nodules, histologic HCC grade, and concomitant liver diseases and subjected tumour tissues to immunohistochemical staining to detect PIVKA-II and AFP expression. The serum PIVKA-II levels of patients with poorly differentiated HCCs were significantly higher than those of patients with well and moderately differentiated HCCs (p<0.05) and they were higher in HCC patients with than without satellite nodules. The serum AFP levels were influenced significantly by concomitant liver diseases, but not by the other factors. Immunohistochemical staining revealed the PIVKA-II expression levels of poorly differentiated HCCs were higher than those of well and moderately differentiated HCCs (p<0.05). Some HCC cells were PIVKA-II-positive, others were AFP-positive, and some expressed both. The serum PIVKA-II concentration was a better indicator of HCC than AFP, as it was not influenced by concomitant liver diseases. The presence of PIVKA-II in serum correlated with the presence of satellite nodules and the histologic HCC grade, a result concordant with the immunohistochemical findings.