A comprehensive collection of cutting-edge methods to study and work with the vaccinia virus and other poxvirus gene transcriptions. These readily reproducible techniques can be used for the construction and characterization of recombinant viruses; for the study of poxvirus gene transcription and DNA replication; for the investigation of the binding, entry, and movement of the virus in host cells; and for the use of the virus in immunologic and cell-cell fusion assays. Other methods cover poxvirus bioinformatics, aspects of viral pathogenesis at both a protein and an animal model level, and the study of immune responses to poxviruses-the latter a critical ability given the important role of vaccinia virus in smallpox vaccination and their potential role as vaccine vectors directed against infectious agents and cancer.