We compared the effectiveness of thyroid transcription factor-1 (TTF-1, cytoplasmic reactivity) and hepatocyte antigen (HPA) as markers for characterization of hepatocellular carcinoma (HCC) and as discriminators to distinguish HCC from its histologic and cytologic mimics. Formalin-fixed, paraffin-embedded sections of 258 specimens, including 76 HCCs, 85 metastatic adenocarcinomas, 75 renal cell carcinomas (RCCs), and 22 adrenal cortical carcinomas (ACCs), were evaluated. Specimens included tissue sections and cytologic material (cell blocks). Following heat-induced epitope retrieval, immunohistochemical studies were performed using an indirect immunoperoxidase technique. Cytoplasmic reactivity for TTF-1 was noted for 54 (71%) of 76 HCCs, 3 (4%) of 85 adenocarcinomas, none of 72 RCCs, and none of 22 ACCs. Cytoplasmic reactivity for HPA was observedfor 50 (66%) of 76 HCCs, 1 (1%) of 83 adenocarcinomas, none of 74 RCCs, and none of 21 ACCs. Cytoplasmic reactivity for TTF-1 and HPA is highly specific for HCC, although a minority of HCCs, particularly poorly differentiated tumors, may be nonreactive. Thus, these markers are usefulfor the characterization of HCC in tissue sections and cell blocks and are highly effective for distinguishing these tumors from other neoplasms included in the differential diagnosis.