Single and double immunohistochemical staining for transforming growth factor (TGF)-alpha and epidermal growth factor-receptor (EGF-R) was done in order to identify the localization of TGF-alpha and EGF-R in human hepatocellular carcinoma (HCC). Single immunohistochemical staining for TGF-alpha showed immunoreactivity in the cytoplasm of hepatoma cells in 22 of 30 cases of HCC. The localization of TGF-alpha was heterogeneous from HCC cells to HCC cells. In the surrounding regenerative nodules, the hepatocytes were mildly to moderately positive for TGF-alpha. The proliferating bile ductules and peripheral nerves were also immunopositive for TGF-alpha. Single immunohistochemical staining for EGF-R demonstrated a linear localization of EGF-R along the cell membrane of the HCC cells in 21 of the 30 cases of HCC. In the regenerative nodules, the hepatocytes also showed linear staining along the cell membrane. Double staining for TGF-alpha and EGF-R in 12 cases of HCC showed a concurrent localization of TGF-alpha and EGF-R in some hepatoma cells and isolated localization of the two substances of other HCC cells. These combinations either abruptly moved around or intermingled with each other. These immonohistochemical results thus support the theory of an autocrine, paracrine, and endocrine mechanism of TGF-alpha and EGF-R on the proliferation of human hepatocellular carcinoma. © 1995 Wiley-Liss, Inc.