Host cell involvement in Sindbis virus (SB) RNA synthesis was examined in cells which had been treated before infection with actinomycin D or alpha-amanitin (alpha-A). Overall synthesis of SB RNA was reduced significantly in CHO cells treated for 18 h before infection with alpha-A. However, SB RNA was produced at near normal levels in CHOama-1 cells, a line which contains an alpha-A-resistant RNA polymerase II. In BHK or CHO cells infected with SBamr, a mutant which replicates normally in cells pretreated with either actinomycin D or alpha-A, viral RNA synthesis was not decreased. The levels of negative strand RNA and of replicative forms I, II, and III in SB-infected cells were progressively reduced with increasing times of pretreatment with host transcription inhibitors, indicating fewer functional replicative intermediates in treated cells. Replicative events after replicative intermediate formation also were inhibited but only to the extent predicted by the reduction in replicative intermediates. Similarly, events preceding negative strand synthesis, adsorption, penetration, uncoating, and translation of nonstructural proteins, apparently were not impeded in treated cells. Therefore, our results are consistent with the involvement of a host component after translation of the nonstructural proteins but before or during the synthesis of SB negative strand RNA.