Sensitivity and specificity of passive hemagglutination (RCA), solid phase radioimmunoassay (RIA), and immunoelectroosmophoresis (IEOP) were compared under experimental and clinical conditions. In dilution experiments with sera containing hepatitis B antigen (HB Ag) of known subtypes, the sensitivity for an ad subtype serum was RIA (1), RCA (1/2), IEOP (1/256) and for an ay subtype serum RCA (1), RIA (1/8), IEOP (1/128). An evaluation of the National Institutes of Health, Division of Biologics Standards test panel number 2 demonstrated HB Ag in 34 of 60 samples by RIA, in 33 by RCA, and in 25 by IEOP. HB Ag was detected in 57.5% of 200 outpatients with a tentative diagnosis of hepatitis by RIA, in 54% by RCA, and in 42.5% by IEOP. In 1,661 volunteer blood donors, 13 (0.78%) were “positive” for HB Ag by RIA, 11 (0.66%) by RCA, and 3 (0.18%) by IEOP. However, absorption experiments indicated that at least six of the above RIA positive and five of the RCA positive sera exhibited nonspecific positive reactions.