Vaccinia virus (VACV) is the live viral component of smallpox vaccine. Inadvertent exposure to VACV can result in infection, and severe complications can occur in persons with underlying risk factors (e.g., pregnancy, immunodeficiencies, or dermatologic conditions). The Advisory Committee on Immunization Practices (ACIP) recommends smallpox vaccination for laboratory workers who handle nonhighly attenuated VACV strains or other orthopoxviruses (e.g., monkeypox, cowpox, or variola). On July 8, 2008, CDC was notified by a Virginia physician of a suspected case of inadvertent autoinoculation and VACV infection in an unvaccinated laboratory worker. This report describes the subsequent investigations conducted by the Virginia Department of Health and CDC to identify the source of infection and any cases of contact transmission. Of the patient's 102 possible contacts, seven had underlying risk factors for developing serious vaccinia infection. Investigators found no evidence of contact transmission and, based on the results of molecular typing, further concluded that the patient had been exposed to a VACV strain that had contaminated the seed stock from the laboratory where the patient worked. This case underscores the importance of adherence to ACIP vaccination recommendations for laboratory workers and use of safety precautions when working with nonhighly attenuated VACV.