Our previous studies employing alpha-amanitin-sensitive H-9 and resistant Ama 102 mutant host cells demonstrated that polymerase II (Pol II), or a drug-sensitive component of the enzyme, is required for replication of vaccinia virus. Evidence was also obtained indicating that transcription from the host genome does not appear to be involved (Silver et al., 1979; Silver and Dales, 1982), suggesting a possible role for Pol II in transcription from the viral genome. This idea is consistent with the present findings, based on immunofluorescence analysis, which revealed that upon infection Pol II antigen is mobilized out of the nucleus into discrete cytoplasmic foci. Effects of treating H-9 rat myoblasts with alpha-amanitin upon vaccinia-specific protein synthesis were also examined. Under the experimental conditions employed, the toxin drastically curtailed in vivo translation into early, late and late-late proteins without altering the spectrum of polypeptides produced. By contrast, treatment with the drug affected, only minimally, the rate of transcription into viral RNA, whether in vivo or from isolated vaccinia factories. The mRNA isolated from infected and treated or untreated cells was translated in a reticulocyte lysate with equal efficiency and general fidelity. This finding suggests that Pol II may be involved in transcription into RNAs related to factors controlling the in vivo translation process. The possible mechanisms for exercising such controls are discussed in relation to factors regulating transcription by host RNA polymerases from a viral DNA genome.