Seventeen ts mutants of vaccinia virus known to map to the viral HindIII D fragment (R. C. Condit and A. Motyczka, 1981, Virology 113, 224-241; R. C. Condit, A. Motyczka, and G. Spizz, 1983, Virology 128, 429-443; M. J. Ensinger and M. Rovinsky, 1983, J. Virol. 48, 419-428) have been sorted into seven complementation groups. The precise location of each mutant on the HindIII D DNA fragment has been identified by either one-step or two-step marker rescue. By a comparison of this genetic map and the known sequence of this DNA fragment (E. G. Niles et al., 1986, Virology 153, 96-112; S. L. Weinrich and D. E. Hruby, 1986, Nucleic Acids Res. 14, 3003-3016), each mutant has been assigned to a single gene in the HindIII D fragment. In several cases, the map position of a mutant has been localized to a region of fewer than 300 bp in length. The complementation groups are evenly distributed along the DNA. However, within a single gene, the mutants are often clustered.