Abstract Thirty-nine new temperature-sensitive mutants of vaccinia virus have been isolated, expanding a previously reported collection of mutants ( R. C. Condit and A. Motyczka, Virology 113, 224–241, 1981 ) to a total of 65. The 65 mutants have been assigned to 32 complementation groups, based primarily on a qualitative spot test described previously ( Condit and Motyczka, 1981). Representatives of each complementation group have been assayed for DNA and protein synthesis at the nonpermissive temperature, revealing one new DNA-negative complementation group, three new groups which contain mutants defective in late protein synthesis, and ten new groups containing mutants which synthesize DNA and protein in a normal fashion. Marker rescue has been achieved with 29 of the 65 mutants using cloned DNA fragments from wild-type virus. These 29 mutants together represent 20 of the 32 complementation groups. A preliminary physical map of the mutants is presented.