We have carried out detailed phenotypic characterization of five temperature-sensitive (ts) mutants of vaccinia virus, the ts lesions of which have previously been mapped to two different subunits of the viral RNA polymerase. We have also attempted to determine the mechanism of temperature sensitivity in these mutants. Phenotypic characterization of each of the mutants showed that at the nonpermissive temperature, all five mutants exhibited normal levels of early viral mRNA and protein synthesis, but for an extended period of time, all mutants accumulated normal levels of DNA in abnormally large pools in the cell cytoplasm; all mutants were defective in the synthesis of late viral mRNA and proteins and in viral morphogenesis. In an attempt to address the mechanism of temperature sensitivity in these mutants, we measured the effect of a temperature shift on the ability of the mutants to direct late viral protein synthesis. If infected cells were shifted down from a nonpermissive temperature late during infection, late protein synthesis was initiated after a lag period of 1 to 2 h. If infected cells were shifted up from a permissive temperature early during infection, late protein synthesis continued to be defective. If infected cells were shifted up to the nonpermissive temperature after late protein synthesis had commenced, late protein synthesis was maintained at the nonpermissive temperature at the level observed when the temperature was shifted up. We interpret these results to mean that once a functional RNA polymerase has been assembled at the permissive temperature during a mutant infection, it remains functional at the nonpermissive temperature, but that the ts mutants are defective in the assembly of a newly synthesized RNA polymerase at the nonpermissive temperature. This interpretation implies that the virion RNA polymerase is responsible for early viral transcription and that a newly synthesized RNA polymerase transcribes late viral genes.