Fibrolamellar carcinoma is a rare malignant primary liver neoplasm with characteristic histological features that typically arises in young patients without viral hepatitis or cirrhosis. Previous studies on this entity have been limited by small numbers of patients. In contrast to classical hepatocellular carcinoma, individual cases of fibrolamellar carcinoma have been reported to express cytokeratin 7. In addition, ultrastructural and serological studies have suggested that fibrolamellar carcinoma may show neuroendocrine differentiation. The cellular differentiation of fibrolamellar carcinoma has not been studied and little is reported about its immunohistochemical profile. We studied 26 cases of fibrolamellar carcinoma and 62 cases of classical hepatocellular carcinoma by immunohistochemistry for HepPar1, glypican-3, pCEA, CD10, alpha-fetoprotein, cytokeratin 20, neuroendocrine markers, and surrogate markers for biliary differentiation (cytokeratin 7, cytokeratin 19, epithelial membrane antigen, EpCAM, mCEA, B72.3, and CA19.9). In situ hybridization for albumin mRNA was also performed. Tumor cells of fibrolamellar carcinoma and hepatocellular carcinoma showed positive signals for albumin mRNA by in situ hybridization in all cases. Both tumor types stained uniformly positively with HepPar1 and most showed a canalicular staining pattern for pCEA, confirming their hepatocellular differentiation. In addition, 39% of hepatocellular carcinoma cases and 59% of fibrolamellar carcinoma cases were positive for glypican-3. All 22 fibrolamellar carcinoma cases tested showed positive staining for cytokeratin 7 and epithelial membrane antigen, whereas less than one-third of hepatocellular carcinoma cases were positive for these markers (P<0.0001). Further, 36% of fibrolamellar carcinoma cases showed staining for B72.3, cytokeratin 19, EpCAM, or mCEA. Minimal evidence of neuroendocrine differentiation in either tumor was found with any of the usual immunohistochemical markers used for this purpose. Therefore, cytokeratin 7 and epithelial membrane antigen may be useful to differentiate between fibrolamellar carcinoma and hepatocellular carcinoma. On the basis of immunohistochemistry, fibrolamellar carcinoma seems to show both hepatocellular and bile duct differentiation.