When our stock of standard Sindbis virus (SVSTD) is assayed by plaque formation on Aedes albopictus mosquito cells, about 1-2% of the plaques appear much clearer and sharper than the majority of the plaques. One of these clear plaques was picked, grown into a viral stock (SVCP), and used to prepare viral cDNA. Making use of the infectious Sindbis virus plasmid, Toto 1101 (Rice et al., 1987), we mapped the causal mutation for the clear plaque phenotype to a region between nt 7334 and 7716, and by sequencing of the viral RNA identified a mutation at nucleotide 7592. This mutation lies in the junction region of the viral genome, specifically at nucleotide -6, with reference to the initiation site for 26 S RNA synthesis. In SVCP-infected mosquito cells, but not in SVCP-infected chick cells, the ratio of subgenomic 26 S to 49 S (genomic) RNA synthesis was decreased relative to that observed in SVSTD infected cells. In terms of amino acid coding, the SVCP mutation is silent.