A cDNA clone prepared from hepatitis delta virus (HDV) RNA extracted from human serum was subcloned in the bacterial expression vector pPL31 to produce a fusion protein consisting of the first 98 amino acids of MS2 polymerase and of 64 amino acids from near the N-terminal region of hepatitis delta antigen (HDAg). The fusion protein was shown to be related to HDAg by a commercial sandwich immunoassay (Abbott) and immunoblotting with human anti-HDAg serum. Antiserum against the fusion protein was raised in rabbits and used to identify HDAg extracted from the serum and liver of an HDV-infected woodchuck and chimpanzee and from the serum of an HDV-infected human, by immunoblotting and immunohistology. A single, major polypeptide of 24K was detected in both serum and liver extracts, with a minor polypeptide of 26K sometimes present. Liver extracts also contained lower Mr polypeptides thought to be degradation products, the major species being 22.5K. The same pattern of staining was obtained with human anti-HDAg serum. Absorption experiments with the expressed protein and cross-competition experiments with the rabbit antiserum suggest that a major immunodominant region of HDAg is present near the N-terminal end of the antigen, between positions 1561 and 1368 on the genome. Both the expressed protein and rabbit antiserum were shown to be good diagnostic reagents.