We have examined the synthesis and processing of nonstructural polyproteins by several temperature-sensitive mutants of Sindbis virus, representing the four known RNA-minus complementation groups. Four mutants that possess mutations in the C-terminal domain of nonstructural protein nsP2 all demonstrated aberrant processing patterns when cells infected with these mutants were shifted from a permissive (30 degrees) to a nonpermissive (40 degrees) temperature. Mutants ts17, ts18, and ts24 showed severe defects in processing of nonstructural polyproteins at 40 degrees, whereas ts7 showed only a minor defect. In each case, cleavage of the bond between nsP2 and nsP3 was greatly reduced whereas cleavage between nsP1 and nsP2 occurred almost normally, giving rise to a set of polyprotein precursors not seen in wild-type-infected cells at this stage of infection. The nsP1 produced by these mutants was unstable and only small amounts could be detected in infected cells at the nonpermissive temperature. Submolar quantities of nsP2 were also present. We suggest that nsP1 and nsP2 may function as a complex and that free nsP1, and possibly nsP2, is degraded. Cleavage between nsP3 and nsP4 appeared to be normal in the mutants except in the case of ts17, where upon shift to 40 degrees P34 was unstable and nsP4 accumulated. We propose that the change in the P34/nsP4 ratio upon shift is responsible for the previously observed temperature sensitivity of subgenomic 26 S RNA synthesis in ts17 and for the failure of the mutant to regulate minus strand synthesis at 40 degrees. Other mutations tested, including ts21, which is found in the N-terminal half of nsP2, ts11, which has a mutation in nsP1, and ts6, which has a mutation in nsP4, all demonstrated nonstructural polyprotein processing indistinguishable from that in wild-type-infected cells. These results support our conclusion, based upon deletion mapping studies, that the C-terminal domain of nsP2 contains the nonstructural proteinase activity.