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Background: Laboratory health care workers (HCWs) may become infected through their occupation with blood-borne pathogens. The aims of this study were determining the seroprevalence of hepatitis B virus (HBV) and the protection offered by HBV vaccine in medical laboratory HCWs., Materials and Methods: A descriptive cross-sectional study was carried out on 203 employers of clinical laboratories. Participant data were obtained through a questionnaire, and the level of antigens and antibodies were measured by enzyme-linked immunosorbent assay (ELISA)., Results: All of the subjects were negative for HBV infection. Forty-seven (23.2%) were not immune, 126 (62.0%) were relatively immune, and 30 (14.8%) were highly immune., Conclusion: Hepatitis B infection is infrequent in laboratory HCWs in Isfahan.