We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia coli β-galactosidase (βgal) gene into the thymidine kinase (TK) locus of the vaccinia virus genome. Tissue culture cells that had been infected with vaccinia virus were transfected with a plasmid vector containing a foreign gene. TK⁻ recombinants could be selected by a plaque assay on TK⁻ cells in the presence of 5-bromodeoxyuridine and distinguished from spontaneous TK⁻ mutants by the addition of a β-gal indicator to the agarose overlay. Plaques that expressed β-gal stained dark blue within several hours at 37°C. Alternatively, TK⁻ selection could be eliminated, and recombinant plaques could be readily identified solely by their blue color. The reverse procedure, in which the starting virus expresses β-gal (i.e., forms blue plaques) and the desired recombinant has deleted the entire β-gal gene (i.e., forms white plaques), is another alternative. Each protocol was tested by constructing vaccinia virus recombinants that express hepatitis B virus surface antigen.