BACKGROUND Hepatitis B infection (HBV) is a significant hazard in the dental environment because the virus may be transmitted through contaminated dental instruments. OBJECTIVE This study determined whether cold disinfectants can inactivate HBV DNA and HBV surface antigens on diamond burs contaminated with HBV and whether ultrasonication can increase the antiviral properties of these agents. METHODS Sterile dental diamond burs were contaminated with serum from a patient who tested positive for HBV surface antigen and hepatitis B viral DNA. The burs were air dried and placed in solutions containing either Cidex, Asepsys, TBS, Rotagerm, Virkon disinfectants, or a control phosphate buffered saline. Burs were divided into 2 groups and disinfected for 15 minutes. The first group was ultrasonicated; the second group was not ultrasonicated during disinfection. All the burs were transferred to phosphate buffered saline and ultrasonicated to remove any remaining viral particles. The ultrasonicate was tested for the presence of HBV surface antigen with a microparticle enzyme immunoassay and for hepatitis B viral DNA with a chemiluminescent molecular hybridization assay. RESULTS TBS did not require ultrasonication to inactivate viral DNA and surface antigen. Rotagerm and Virkon inactivated surface antigen and viral DNA only with ultrasonication. Cidex and Asepsys inactivated viral DNA but not surface antigen with ultrasonication. CONCLUSIONS The chlorine containing compound TBS was the most active disinfectant tested and did not require ultrasonication to destroy HBV. The remaining disinfectants should be used with ultrasonication to inactivate HBV.