Expression of the epidermal growth factor (EGF), EGF receptor (EGF-R) and ras oncogene product p21 was simultaneously examined in 37 cases with intrahepatic cholangiocarcinoma (CC) by means of an immunocytochemical method. While normal livers were all negative for any of the antigens at the concentration of the antibodies used, EGF-R was positive in 12 (32.4%) CCs, EGF in 22 (59.5%), and ras p21 in 33 (89.2%). The positive incidence of the three antigens was not different among the histologic subtypes of the tumor. However, the number of EGF-R- and ras p21-positive tumor cells decreased with progressing histologic tumor grade, but the expression of EGF was not associated with the tumor grade. Expression of the three antigens was not related to the degree of metastatic spread of the tumor. Simultaneous expression of the three antigens was seen only in 4 CCs, and that of EGF-R and EGF in 4, EGF-R and ras p21 in 12, and EGF and ras p21 in 20. These data suggest that the expression of EGF, EGF-R and ras p21 on CC cells is not related to the tumor aggressiveness, and the activation of each respective gene is independent. Furthermore, the data also indicate that an autocrine model for tumor growth, as suggested by a combination of EGF and EGF-R, may be applicable only to very limited cases of CCs.