RNA was synthesized in nuclei isolated at various times postinfection from the fat bodies of Heliothis zea larvae infected with H. zea nuclear polyhedrosis virus and from cultured Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus. To detect virus-specific RNA synthesis, the [3H]RNA was hybridized to denatured viral DNA immobilized on nitrocellulose filters. Nuclear polyhedrosis virus-specific RNA synthesis in the infected nuclei isolated from H. zea larval fat bodies and S. frugiperda cells was only inhibited 20 to 25% by concentrations of a-amanitin sufficient to inhibit the host RNA polymerase II. In addition, a productive nuclear polyhedrosis virus infection was obtained in S. frugiperda cells grown in the presence of an a-amanitin concentration that inhibited 90% of the cellular RNA polymerase II activity. The cellular RNA polymerase II enzyme remained sensitive to a-amanitin during infection, and there was no evidence that a virus-coded, a-amanitin-resistant enzyme was synthesized after the onset of infection. The data suggest that the bulk of nuclear polyhedrosis virus-specific RNA synthesis in isolated nuclei is transcribed by an enzyme other than the host RNA polymerase II.