Vaccinia virus, the prototype Orthopoxvirus, is widely used in the laboratory as a model system to study various aspects of viral biology, virus–host interactions, and as a protein expression system and a vaccine vector. The ubiquitous use of vaccinia viruses 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 vaccinia virus and issues surrounding the use of prophylactic smallpox vaccination for laboratory workers. Key Words: Vaccinia virus; biosafety level 2; class II biological safety cabinet; personal protective equipment; smallpox vaccine; vaccination complications.