Host cell involvement in Sindbis virus (SB) replication was examined in cells which had been treated with either actinomycin D (AMD) or alpha-amanitin (alpha-A). Treatment with these inhibitors of host transcription before infection reduced the ability of cells to support SB growth by 1 to 2 orders of magnitude, while having little or no effect on the replication of vesicular stomatitis virus. SB replication was sensitive to alpha-A in wild-type Chinese hamster ovary (CHO) cells but was resistant to alpha-A in CHOama-1 cells, a line which contains an alpha-A-resistant RNA polymerase II. A mutant of SB, SBamr, was isolated by mutagenesis followed by selection in cells which had been treated with AMD. SBamr grew normally not only in cells treated with AMD but also in alpha-A-treated cells. Our results suggest (i) that the synthesis of cellular mRNA (and presumably protein) is required for replication of SB, (ii) that prior treatment with either drug affects the same aspect of SB replication, and (iii) that mutations in the SB genome allow the virus to overcome the effect of inhibitors of host transcription.