The inhibition of ribosomal RNA production has been studied in Hela cells infected with pseudorabies virus (PrV). This showed that both synthesis and processing of ribosomal RNA was affected. Processing in PrV-infected Hela cells was at a reduced rate but did follow a similar pattern to that of mock-infected cells. The effect on synthesis of ribosomal RNA was more pronounced and was followed by a concomitant decrease in the total nucleolar area of the infected Hela cells. Polyacrylamide gel electrophoresis of fractions from mock-infected and PrV-infected Hela cells showed that a number of virus induced polypeptides were associated with the nucleus, nucleolus and the nucleolar chromatin. Some of these polypeptides were immediate early viral gene products. A number of viral induced polypeptides were shown to be associated with the nuclei and nucleoli of mock-infected Hela cells after incubation in vitro of isolated nuclei and cell extracts of both mock-infected and infected Hela cells. The RNA polymerase activity of isolated nuclei of PrV- infected Hela cells was lower than that of mock-infected. This was true both for total and for a-amanitin resistant activities. The difference in RNA polymerase activity between mock-infected and infected Hela cells remained unchanged after incubation of the nuclei in the presence of the detergent sarkosyl. Again incubation in the presence of extracts of mock-infected or infected cells did not alter relative activities in the normal assay whereas RMA polymerase activity in mock-infected Hela cell nuclei was reduced after a combined exposure to infected cell extracts followed by treatment with aarkosyl. This suggests that its inhibition of ribosomal RNA synthesis in Hela cells after infection with pseudorabies virus is by interference at the initiation level of the transcription process rather than the elongation level. It may be mediated by a viral-induced protein(s) associated with the nucleolar and nucleolar chromatin cell fractions.