Previous studies have demonstrated an elevated level of heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA in hepatocarcinogenesis in a rodent model and of its protein as well as mRNA in human hepatocellular carcinoma (HCC). In order to elucidate the clinical significance of HB-EGF in human HCC, we immunohistochemically investigated its expression in 100 HCC cases with different characteristics. HB-EGF was not or only faintly expressed in non-cancerous hepatocytes. On the other hand, 59% of the HCC cases were classified as positive for HB-EGF, and its expression was more frequently observed in cases with low proliferating activity, at an early stage, of small size, without portal invasion, low alpha-fetoprotein level, without satellite nodules (borderline significance), and well or moderate differentiation. Furthermore, HB-EGF expression showed prognostic significance in both univariate and multivariate analyses and can be regarded as an independent prognostic factor of disease-free survival. These results strongly suggest the involvement of HB-EGF in early stage of HCC.