Template specificity of two RNA-dependent RNA polymerases (Qbeta and SP RNA replicases) was examined using "variant RNAs" as template. Three variant RNAs, one (8S) generated by Qbeta replicase and two (6S and 5.2S) generated by SP replicase, were isolated from the reaction mixtures incubated in the absence of exogenous template RNA. All these RNAs were found to be active as template for both Qbeta and SP replicases, though homologous RNA exhibited activities about three times higher than heterologous RNA with either enzyme, in agreement with the results obtained in phage RNA-dependent reactions. In these reactions, faithful replication of variant RNA was observed, and the amount of RNA synthesized was in a many-fold excess over the template RNA added. We also found that the heterologous RNA-dependent reactions were suppressed by increasing the concentration of salts or decreasing the concentration of substrates. Under such conditions, replication of heterologous variant RNA was almost completely suppressed, while the amount of homologous variant RNA synthesized was only reduced to 50% of that synthesized under the standard conditions. Thus the template specificity of the two RNA replicases seems to be expressed more strictly in these replication systems.