We present our experience with a new sensitive in vitro method for the study of the immune response to hepatitis B surface antigen (HBsAg). This immunoenzymatic technique, called spot-ELISA, detects specific immunoglobulin production (e.g. IgG- or IgM-anti-HBs) by stimulated individual B cells in vitro. We have studied the immune response against HBsAg by mitogenic stimulation and subsequent spot-ELISA assay in 24 well-documented subjects with known immune status after either natural infection or vaccination with plasma-derived vaccine. Results indicate a good correlation between in vitro IgG anti-HBs spots and immune 'memory.' A possible predictive value in non-immunized and non-responder subjects awaits further confirmation.