Two positive-strand mRNAs are made in Sindbis virus-infected cells, the genomic (G) RNA and the subgenomic (SG) RNA. In mosquito cells infected with wild-type (wt) Sindbis virus, the latter is made in excess over the former; however, in cells infected with SVpzf or SVcpc more G RNA is made than SG RNA. Use was made of in vitro systems to investigate the effects of the SVpzf and SVcpc mutations on the synthesis of SG and G RNAs. Our findings indicate that under standard reaction conditions, the SG/G RNA ratio in vitro reflects the ratio of SG to G RNA made in infected mosquito cells. We observed further that the RNA patterns seen in vitro are affected not only by the SVpzf and SVcpc mutations but also by the nucleoside triphosphate concentrations in the reaction mixtures and that introduction of these mutations into nsP4 and the promoter/template change the relative amounts of SG and G RNAs that are made, likely through the choice of promoter. We conclude that with respect to the SVpzf and SVcpc mutations, it is mainly the nucleotide changes in the SG promoter, not the amino acid changes in nsP4, that determine the SG/G RNA ratio that results. Further, it was observed that the SVpzf mutations enhance the in vitro synthesis of SG RNA at the lowest concentrations of UTP/CTP and that the single SVcpc mutation enhances the synthesis of G RNA at the lowest concentrations of CTP tested. We also identified three Arg residues in nsP4, R545, R546, and R547, that are needed for the synthesis of G RNA but not SG RNA.