We have studied the expression pattern of the vaccinia virus DNA polymerase during the viral replicative cycle. To monitor polymerase synthesis, a polyclonal antiserum was raised against a TrpE-DNA polymerase fusion protein. Immunoprecipitation and S1 analyses revealed that polymerase synthesis and mRNA levels peak by 2 to 3.5 h postinfection during wild-type infections and then decline, becoming barely detectable by 5 to 6.5 h postinfection. Blocking viral DNA replication by performing infections with temperature-sensitive DNA- mutants at the nonpermissive temperature or by performing wild-type infections in the presence of cytosine beta-D-arabinofuranoside had no effect on polymerase expression. These results indicate that the transient expression of the DNA polymerase is regulated independently of intermediate and late viral gene expression. Cycloheximide, which inhibits protein synthesis and prevents secondary uncoating, caused prolonged and elevated levels of polymerase transcription. Early viral proteins and uncoating, rather than exhaustion of the encapsidated transcription machinery, are presumed to mediate the cessation of polymerase transcription. In the presence of aphidicolin, the polymerase transcripts were maintained at maximal levels rather than exhibiting their normal decline. This inhibition of RNA decay was seen even in infections performed with isolates encoding aphidicolin-resistant DNA polymerases, suggesting that aphidicolin may interfere directly with the process of RNA degradation. Under these conditions, polymerase synthesis remained transient and was not prolonged, despite the continuing presence of available mRNA. These observations suggest that early mRNAs may experience a loss in translation efficiency as infection progresses.