OBJECTIVE Polymerase chain reaction (PCR) is very sensitive. The aim of the study was to reevaluate viral replication in hepatitis D virus (HDV) superinfection by PCR. METHODS HDV and hepatitis B virus (HBV) were detected by PCR in 185 patients. RESULTS The acute hepatitis group had the highest detection rate of HDV RNA compared with chronic hepatitis, cirrhosis, hepatocellular carcinoma, and remission groups (63 of 64 vs. 35 of 47, 17 of 23, 19 of 30, and 7 of 21) and the highest alanine aminotransferase (ALT) levels (mean, 1741 U/L vs. 266 to 27 U/L; P < 0.05). The detection rate of HBV DNA was the lowest in the acute group (41%) compared with 66%, 70%, 80%, and 57% in the remaining groups (P < 0.02). At the chronic stage, 13%-25% of cases had HDV RNA, and 30%-48% of cases had HBV DNA detected by PCR but not by traditional method. HDV RNA was associated with ALT levels in horizontal and longitudinal analyses. CONCLUSIONS HDV superinfection may be divided into the following three phases: acute phase, active HDV replication and suppression of HBV with high ALT levels; chronic phase, decreasing HDV and reactivating HBV with moderate ALT levels; and late phase, development of cirrhosis and hepatocellular carcinoma caused by replication of either virus or remission resulting from marked reduction of both viruses.