The Sindbis virus (SIN) nonstructural protein nsP4 possesses the RNA-dependent RNA polymerase activity required for the replication of the SIN genome and transcription of a subgenomic mRNA during infection. Isolation of this protein from other viral components of the RNA synthetic complex allowed the characterization of template requirements for nsP4-mediated genome replication. The major findings of this study are: (i) in the absence of other viral proteins nsP4 is capable of copying SIN plus- and minus-strand templates, but does not transcribe subgenomic RNA; (ii) mutations in the 3' conserved sequence element and poly(A) tail of the plus-strand template prevent nsP4-mediated de novo initiation of minus-strand RNA synthesis; (iii) nsP4-dependent terminal addition of nucleotides occurs on template RNA possessing certain mutations in the 3'CSE and polyadenylate tail ; (iv) nsP4 is capable of minus-strand synthesis independent of the sequence at the 5' end of the template; (v) an A-U rich sequence in the 3'CSE represents a binding site for a replicase component, probably nsP4; (vi) plus-strand genomic RNA synthesis is dependent on the 3' end of the minus-strand template. These studies begin to define the specific interactions with the viral RNA templates mediated by individual components of the viral replication complex and suggest a model for ternary complex formation during the initiation of minus-strand RNA synthesis.