A transient expression system has been developed to compare the relative efficiency of expression of various vaccinia virus DNA sequences containing transcriptional regulatory elements. A plasmid vector was constructed containing both the Escherichia coli galactokinase gene (galK) and the guanine phosphoribosyltransferase gene (gpt). To direct the expression of gpt within this vector, a vaccinia virus promoter region was isolated from the HindIII-F fragment of the genome and inserted 5' to gpt coding sequence. Four unique cloning sites in front of galK allow simple and precise fusion of various vaccinia virus DNA fragments that contain the regulatory site of interest to galK. Sequences containing promoter regions were ligated to the coding segment of the galK to create four recombinant plasmids, which were introduced into vaccinia virus-infected cells by transfection. Both galK and gpt were thus expressed under the control of vaccinia virus transcriptional units, and the enzymatic activities were measured in the same cell extract with a filter-binding assay. The major advantage of this transient expression system is that the variations in galK expression are always measured relative to the internal gpt standard. Changes in the galK/gpt ratio resulting from different vaccinia promoters of galK are thus a quantitative measurement of promoter strength.