Vaccinia virus (VACV), the prototype orthopoxvirus, is widely used in the laboratory as a model system to study various aspects of viral biology and virus–host interactions, as a protein expression system, as a vaccine vector, and as an oncolytic agent. The ubiquitous use of VACVs in the laboratory raises certain safety concerns because the virus can be a pathogen in individuals with immunological and dermatological abnormalities, and on occasion can cause serious problems in normal hosts. This chapter reviews standard operating procedures when working with VACV and reviews published cases on accidental laboratory infections.