A heterologous system expressing functional Sindbis virus nonstructural proteins (nsPs) has several possible uses for studying Sindbis virus-specific RNA replication and transcription in vivo and in vitro. Of the many possible approaches, vaccinia virus offers an attractive transient expression system given that Sindbis virus replication can occur in cells which have been previously infected by vaccinia virus. In this report, a vaccinia virus recombinant (called vSINNS), which contains the cDNA encoding the Sindbis virus nsPs under the control of either the vaccinia virus 7.5K promoter or the bacteriophage T7 promoter, has been constructed and characterized. Upon infection of several cell types with vSINNS, Sindbis virus nsP precursors and processed forms, including nsP1, nsP2, and both phosphorylated and nonphosphorylated forms of nsP3, were synthesized. Proteins containing the putative RNA-dependent RNA polymerase domain (nsP4 and nsP34), which are normally produced in small amounts by readthrough of an opal termination codon, were not detected in vSINNS-infected cells. However, all nsP functions necessary for Sindbis virus-specific RNA synthesis must have been expressed, since both replication and subgenomic mRNA transcription of an engineered Sindbis virus defective interfering RNA in cells infected with vSINNS was observed. Furthermore, vSINNS could be used as a helper virus to amplify, to relatively high titers, a replication-defective Sindbis virus mutant containing an in-frame deletion in the conserved N-terminal domain of nsP3. These data, as well as the observation that normal yields of parental Sindbis virus are produced in cells which have been previously infected with vSINNS, indicate that expression of Sindbis virus nsPs, in the absence of Sindbis virus-specific RNA replication, is not sufficient to block the formation of active RNA replication complexes by superinfecting Sindbis virus.