We assessed the significance of the enhanced detection by monoclonal radioimmunoassay (M-RIA) of HBsAg in serum of patients with hepatitis B virus (HBV) infection. In acute HBV infection, the M-RIA detected HBsAg in the blood for a far longer period of time than previously recognized. In some patients, the “window phase” of HBV infection (defined as the presence of anti-HBc and the lack of detectable HBsAg and anti-HBs) was shortened or completely eliminated. Furthermore, 26% of individuals with anti-HBc as the only serologic marker of recent or past HBV infection were reactive only in the M-RIA. Analysis of patients tested for HBsAg because of suspected underlying liver disease revealed additional HBsAg-positive sera, unrecognized by polyvalent RIA, thus adding important information in the immunodiagnosis of HBV infection. The enhanced performance of the monoclonal RIA compared to conventional polyvalent RIA was due in part to the increased sensitivity of the assay and the observation that M-RIA may detect “hidden” HBV antigens in immune complexes.