Current knowledge on the expression of HBeAg in hepatocytes is incomplete because of difficulties in obtaining monospecific antisera devoid of anti-HBc reactivity. In this study, we have examined by immunofluorescence the expression of HBcAg and HBeAg in cryostat liver sections from 25 chronic carriers of HBsAg using monoclonal antibodies. Although virtually all liver biopsies displayed concordance for HBeAg and HBcAg expression, the pattern of fluorescence differed markedly. Thus, monoclonal anti-HBc gave nuclear staining in all 13 reactive biopsies, while cytoplasmic staining was observed in only two of these. In contrast, monoclonal anti-HBe showed cytoplasmic reactivity coexisting with nuclear reactivity in 10 of 13 reactive biopsies. Hepatitis B virus DNA polymerase activity in the serum appeared to correlate better with the presence of HBcAg in hepatocytes rather than HBeAg. These results provide further evidence that HBeAg is expressed both in the nuclei and in the cytoplasm of infected hepatocytes. The observation that the number of cells expressing HBeAg exceeds those expressing HBcAg in carriers with active virus replication would suggest that assembly of core particles occurs in only a proportion of infected hepatocytes expressing HBeAg.