We studied the relationship between hepatocyte proliferation and hepatitis delta virus (HDV) replication at the single cell level. The proliferating cell nuclear antigen (PCNA) (by immunohistochemistry) and the HDV RNA (by in situ hybridization) were stained in neoplastic and non-neoplastic liver tissues of 19 patients with chronic HDV infection, including four cases of cirrhosis with superimposed hepatocellular carcinoma (HCC). As controls, we assessed the hepatocyte proliferation of liver tissues from 16 patients with chronic hepatitis B and on three normal livers. The hepatocyte PCNA labelling index of HDV-infected tissues was comparable with that seen in chronic hepatitis B-infected livers but was significantly higher than that observed in normal livers. Although cirrhotic tissues had lower hepatocyte proliferating fractions than non-cirrhotic tissues, the difference was not statistically significant. The hepatocyte proliferation rate did not correlate with the level of intrahepatic HDV replication or with the histological activity. In double-labelling experiments, PCNA and HDV RNA staining did not co-localize, with the exception of two of three cirrhotic tissues associated with HCC, where the association between the two markers was statistically significant. This co-localization was not observed, however, in the adjacent tumorous tissues. In patients with chronic HDV infection the hepatocyte proliferation was increased with respect to normal liver tissue, but was comparable with that observed in patients with chronic hepatitis B virus infection and did not correlate with the level of HDV replication or the histological activity. In the cirrhotic tissue of patients with HCC (but not in the tumour counterpart), HDV RNA may occasionally co-localize with the marker of hepatocyte proliferation. Whether this association between viral replication and cell division is related to liver carcinogenesis remains to be established.