Hepatocyte nuclear factor-4 alpha (HNF4 alpha) exists in multiple isoforms that are generated by alternative promoter (PI and P2) usage and splicing. Here we establish monoclonal antibodies (mAbs) for detecting P1 and P2 promoter-driven HNF4 alpha, and evaluate their expression in normal adult human tissues and surgically resected carcinomas of different origins. Using immunohistochemical analysis, we demonstrate that, while P1 promoter-driven HNF4 alpha is expressed in hepatocytes, small intestine, colon, kidney and epididymis, P2 promoter-driven HNF4 alpha is expressed in bile duct, pancreas, stomach, small intestine, colon and epididymis. Altered expression patterns of PI and P2 promoter-driven HNF4 alpha were observed in gastric, hepatocellular and colorectal carcinomas. HNF4 alpha was expressed in lung metastases from renal cell, hepatocellular and colorectal carcinoma but was not observed in lung tumours. The P1 and P2 promoter-driven HNF4 alpha expression pattern of tumour metastases correlated with the primary site of origin. P1 promoter-driven HNF4 alpha was also found in intestinal metaplasia of the stomach. These data provide evidence for the tissue distribution of P1 and P2 promoter-driven HNF4 alpha at the protein level and suggest that HNF4 alpha may be a novel diagnostic marker for metastases of unknown primary. We propose that the dysregulation of alternative promoter usage of HNF4 alpha is associated with the pathogenesis of certain cancers. Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.