The four nonstructural proteins (nsP1-4) of Sindbis virus, a member of the Togaviridae family, are initially expressed from the 5' segment of the single-stranded genomic (+)RNA as a polyprotein which is subsequently proteolytically processed. In attempts to identify the protease acting on this nonstructural polyprotein, we established a coupled in polyprotein, we established a coupled in vitro transcription-translation system which was able to faithfully process the major polyprotein when an mRNA encoding all four nonstructural proteins was used. A cDNA plasmid containing the entire Sindbis virus genome positioned immediately downstream of the phage SP6 polymerase promoter was cut with restriction endonucleases at sites located within the genes for the nonstructural proteins and mRNAs transcribed from these DNA fragments. The nsP1-2 and nsP2-3 cleavage sites are alanyl-alanine and both were susceptible to proteolysis in vitro only after all of nsp1 and nsP2 and 157 amino acids of nsP3 were translated. The nsP1-2 site was cleaved from a polyprotein that contained nsP1 and nsP2 and 59 amino acids of nsP3 but not from six polyproteins whose sequences terminated in the nsP2 gene. These data support our hypothesis that the nonstructural polyprotein is processed by a virus autoprotease and we propose that its active site is encoded within the nsP2 sequences.