A selective plaque assay that uses thymidine kinase (TK)-deficient human 143 cells was developed to titer mixtures of TK+ and TK- vaccinia virus. With this assay it could be shown that methotrexate-resistant TK+ virus was formed in cells coinfected with TK- virus and wild-type virus DNA. By substituting vaccinia DNA fragments cloned in plasmids for virion DNA, this marker rescue system provided the basis for mapping the TK gene. Of the 15 HindIII fragments, only J could rescue five independently derived TK- mutants. This 5000-base-pair (bp) fragment maps approximately 80,000 bp from the left-end of the 180,000-bp vaccinia genome. Marker rescue could be detected with 18 ng or less of plasmid and was proportionate to DNA concentration. The resistance to methotrexate of the TK+ recombinants was shown to be due to TK synthesis. Evidence that the HindIII J fragment contains the structural TK gene and not a regulatory element was demonstrated by the synthesis of active TK in a cell-free system programmed with mRNA selected by hybridization to the plasmid. Previous studies [Belle-Isle, H., Venkatesan, S. & Moss, B. (1981) Virology 112, 306-317] indicated that mRNAs coding for three immediate early polypeptides with molecular weights of 41,000, 21,000, and 17,000 map within HindIII J. The mapping of the easily selectable vaccinia virus TK gene now opens the way to genetic manipulations that should increase our understanding of vaccinia virus gene expression and facilitate the use of vaccinia virus as an efficient cloning vector for foreign genes.