To investigate the presence of serum hepatitis delta virus antigen by immunoblot and its correlation with other markers of active viral replication (intrahepatic hepatitis D antigen, IgM antibody to hepatitis D and serum hepatitis D virus RNA), we studied serum samples from 50 patients with chronic hepatitis D virus infection (38 with and 12 without intrahepatic hepatitis D antigen). Of the 38 patients with intrahepatic hepatitis D antigen, 27 (71%) had antigen detectable in serum by immunoblot, whereas only two were reactive by conventional enzyme-linked immunosorbent assay. Thirty-one (82%) patients were also positive for serum hepatitis D virus RNA by spot hybridization and 33 (87%) were positive for IgM anti-hepatitis D virus. All markers were simultaneously present in 24 patients. Circulating hepatitis D antigen was detected in one (8%), IgM anti-hepatitis D in seven (58%) and hepatitis D virus RNA in two (17%) of the 12 patients who had anti-hepatitis D in serum but not detectable hepatitis D antigen in liver. Hepatitis D antigen was not detected in serum of any of the 15 control patients. The results suggest that serum hepatitis D antigen as detected by immunoblot and serum hepatitis D virus RNA are similar in sensitivity for detection of active hepatitis D virus replication during chronic infection and constitute useful, sensitive and noninvasive tests for the diagnosis and monitoring of chronic hepatitis D virus infection.