This chapter describes the use of several of the recombinant vaccinia expression systems, focuses on the systems that are most useful for cell biologists, and discusses their advantages and limitations. Vaccinia-mediated expression can be used for assessing cellular localization, posttranslational modifications, oligomerization, and transport and turnover rates. The system provides a rapid method for screening mutant proteins for expression and targeting. It is an excellent way of quickly deciding which mutant proteins might be worth further studying using stable expression systems. Expression of foreign genes using Vaccinia virus is based on recombinant viruses constructed by insertion of complementary DNA (cDNA) into the nonessential thymidine kinase (TK) gene. Both direct and indirect methods of expression are possible. The foreign gene can be inserted into the vaccinia genome by homologous recombination using a plasmid with flanking regions of vaccinia DNA. The recombinant virus is selected, expanded, and used to infect cells, which then express high levels of the foreign protein. Recombinant vaccinia viruses are generated by subcloning the foreign gene into a plasmid transfer vector so it is flanked by DNA from the vaccinia (TK) gene, which is nonessential for growth of the virus in tissue culture. This plasmid is then transfected into vaccinia-infected cells. Homologous recombination of the plasmid and the vaccinia genome generates a recombinant virus with an inactive TK gene.