To determine the need for immunization of health workers with antibody to hepatitis B surface antigen (anti-HBs) as their only serologic marker of previous hepatitis B exposure, we studied the level, persistence, and immunologic specificity of isolated anti-HBs in 46 persons identified during screening for hepatitis B vaccine. We rescreened these persons 1 year later, administered a single dose of hepatitis B vaccine, and determined the anti-HBs level at 1, 2, and 8 weeks after vaccination. Isolated anti-HBs levels were low and antibody did not persist; 22 subjects tested had lost detectable anti-HBs within 19 months even though immunologic specificity was shown in vitro in 34. Anamnestic responses suggesting previous exposure and immunity were seen in only 10 subjects; 5 of these subjects had moderate-level, persistent anti-HBs. Although some persons with naturally acquired, isolated anti-HBs may be protected from hepatitis B, the immunologic specificity and protective value of anti-HBs, especially when levels are low, remain questionable.