Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) viruses (L-A and L-BC) and two different single-stranded (ssRNA) replicons (20S RNA and 23S RNA). Replicase (dsRNA synthesis on a ssRNA template) and transcriptase (ssRNA synthesis on a dsRNA template) activities have been described for L-A and L-BC viruses, but not for 20S or 23S RNA. We report the characterization of a new in vitro RNA replicase activity in S. cerevisiae. This activity is detected after partial purification of a particulate fraction in CsCl gradients where it migrates at the density of free protein. The activity does not require the presence of L-A or L-BC viruses or 23S RNA, and its presence or absence is correlated with the presence or absence of the 20S RNA replicon. Strains lacking both this RNA polymerase activity and 20S RNA acquire this activity when they acquire 20S RNA by cytoduction (cytoplasmic mixing). This polymerase activity converts added ssRNA to dsRNA by synthesis of the complementary strand, but has no specificity for the 3' end or internal template sequence. Although it replicates all tested RNA templates, it has a template size requirement, being unable to replicate templates larger than 1 kb. The replicase makes dsRNA from a ssRNA template, but many single-stranded products due to a terminal transferase activity are also formed. These results suggest that, in contrast to the L-A and L-BC RNA polymerases, dissociation of 20S RNA polymerase from its RNA (or perhaps some cellular factor) makes the enzyme change its specificity.