A turnip yellow mosaic virus RNA-dependent RNA polymerase activity was used to study the template requirements for in vitro minus strand synthesis, which is initiated specifically opposite the 3′-CCA that terminates the 3′-tRNA-like structure. A deletion survey confirmed earlier results suggesting the absence of minus strand promoter elements upstream of the pseudoknotted acceptor stem and 3′-terminus. Reiteration of this 27-nt domain provided two competing initiation sites. By varying the added downstream element, it was shown that the pseudoknotted domain could be functionally replaced by various simple stem/loops, although with some decrease in activity. The addition of varying numbers of consecutive -CCA- triplets to the 3′ end of the tRNA-like structure resulted in accurate initiation from each added triplet. A similar spectrum of initiations occurred with an unstructured RNA consisting of 12 consecutive -CCA- triplets and no additional viral sequence. Substitution mutations revealed no influence on minus strand synthesis of the identity of the nucleotide immediately upstream of a -CC- initiation site, but a preference for a purine immediately downstream. The introduction of secondary structure into the linear template showed that the usage of potential -CCR- initiation sites is influenced by nonspecific secondary structure. We conclude that specificity arises from the requirement that a -CCR- sequence be sterically accessible. This mechanism is only applicable to interactions that do not involve RNA unwinding during site selection, but may be used commonly in positive strand RNA virus replication and be applicable to other RNA–protein interactions.