Replication of the satellite RNA of tobacco ringspot virus (sTobRV RNA) has been postulated to require rolling circle transcription. The expected product of rolling circle transcription, multimeric sTobRV RNA, is known to undergo self-cleavage in vitro to release unit-length sTobRV RNA. A spontaneous, efficient, not enzymically-catalyzed in vitro circularization reaction is characteristic of unit-length sTobRV RNA of the less abundant, (-) polarity. We mutated sTobRV RNA at two sites that are distant from each other in the polyribonucleotide chain. A third form of the sTobRV RNA was mutated at both sites. Multimeric forms of the one-site mutants of sTobRV(+)RNA and sTobRV(-)RNA showed, respectively, undiminished and slightly diminished self-cleavage, whereas the spontaneous circularization of each one-site-mutated, unit-length sTobRV(-)RNA was greatly reduced, compared to the reactions of wild-type sTobRV RNA and the two-site mutant. The two-site mutant and the wild-type sTobRV RNAs replicated with similar efficiency. They reduced the titer of, and severity of, symptoms induced by coinoculated tobacco ringspot virus (TobRV). When coinoculated with TobRV, neither one-site mutant increased or provided protection against TobRV. Rather, each induced a substantial accumulation of what is apparently an endogenous form of sTobRV RNA. Our results are consistent with the formation of circular sTobRV(-)RNA as an essential step in sTobRV RNA replication.