Alphaviruses are alternately transmitted between arthropod and vertebrate hosts. In each host, the virus transcribes a subgenomic mRNA that encodes the viral structural proteins which encapsidate the genome to form progeny virions. Transcription initiates at an internal site called the promoter. To determine if promoter utilization varies in mammalian versus mosquito cells, we used these cells as hosts to select for active promoters among a library of different mutant promoters. Compared with that in BHK-21 cells, selection was more rapid in mosquito (C7-10) cells, with much less diversity of promoters remaining after fewer passages. Thus, promoter selection is host dependent. With further passaging, both BHK-21 and C7-10 cells selected for similar sequences that closely resemble the wild-type promoter sequence. The difference in the rates of selection is not because BHK-21-derived promoters cannot function in mosquito cells. Instead, part of the host dependence is probably due to posttranscriptional differences between BHK-21 and C7-10 cells that may require more active promoters in mosquito cells. Part of the host dependence may also be attributed to the decreased rate of transcription versus that of replication in mosquito cells. This change in regulation of subgenomic to genomic RNA synthesis appears to correlate with the extent of cleavage or pausing of the genomic RNA synthesis at or close to the promoter.