The time sequence, relative reactivity, and persistence of anti-HBc IgM were assessed in patients with HBsAg-positive viral hepatitis. A solid-phase immunoassay was developed using the IgM capture procedure with anti-mu-coated polystyrene beads. HBcAg was purified from serum Dane particles and used as a probe with 125I-labeled anti-HBc IgG. This immunoassay exhibited a pronounced prozoning phenomenon, and relative titers of sera differed widely depending upon the dilution of serum tested. When all sera were tested at 1:5,000 dilution, results were comparable in different patient groups. Anti-HBc IgM persisted at detectable levels for up to 2 years, and it was necessary to establish relative titers to discriminate current from remote infections. A cut-off assay value was established, and in 12 cases of acute hepatitis B virus (HBV) infection, antibody exceeded this value for about 6 months after onset of HBs antigenemia. A similar profile of anti-HBc IgM persistence was observed in seven patients who developed an HBsAg chronic carrier state. Long-term viral replication did not sustain elevated IgM class-specific antibody levels. The studies suggest that anti-HBc IgM analyses may be useful for differentiating recent and current HBV infections from remote infections, eliminating HBV as the agent for non-A, non-B hepatitis in asymptomatic HBsAg carriers, and detecting HBV as the etiologic agent during silent (HBsAg negative) infections.