The Sindbis virus minimal subgenomic mRNA promoter (spanning positions 19 to +5 relative to the subgenomic mRNA start site) is approximately three- to sixfold less active than the fully active 98 to +14 promoter region. We identified two elements flanking the 19 to +5 region which increase its transcription to levels comparable to the 98 to +14 region. These elements span positions 40 to 20 and +6 to +14 and act synergistically to enhance transcription. Nine different virus libraries were constructed containing blocks of five randomized nucleotides at various positions in the 40 to +14 region. On passaging these libraries in mosquito cells, a small subset of the viruses came to dominate the population. Sequence analysis at the population level and for individual clones revealed that in general, wild-type bases were preferred for positions 15 to +5 of the minimal promoter. Base mutagenesis experiments indicated that the selection of wild-type bases in this region was primarily due to requirements for subgenomic mRNA transcription. Outside of the minimal promoter, the 35 to 29 region contained four positions which also preferred wildtype bases. However, the remaining positions generally preferred non-wild-type bases. On passaging of the virus libraries on hamster cells, the 15 to +5 region again preferred the wild-type base but most of the remaining positions exhibited almost no base preference. The promoter thus consists of an essential central region from 15 to +5 and discrete flanking sites that render it fully active, depending on the host environment.