Most strains of Saccharomyces cerevisiae harbor L-A double-stranded RNA (dsRNA), 4.5 kilobases long, contained in virus-like particles (VLPs). These L-A VLPs can be separated by CsCl density gradient centrifugation into a main peak of particles, containing full-length L-A dsRNA, which synthesizes only plus-strand single-stranded RNA (ssRNA), and a lighter fraction of VLPs, containing plus-strand ssRNA, which has L-A dsRNA-synthesizing activity. This dsRNA-synthesizing activity was present in particles from logarithmically growing cells but not from stationary-phase cells. The newly synthesized strand of dsRNA in the lightest particles was full-length minus strand. All or almost all of the new minus strand was synthesized in vitro, and the rate of chain elongation was approximately 100 nucleotides per minute. The lightest particles synthesized plus-strand ssRNA only after completion of dsRNA synthesis, indicating that the same particle contains dsRNA- and ssRNA-synthesizing enzyme(s). We also observed dsRNA-synthesizing activity in L-BC dsRNA-containing particles similar to that in L-A VLPs.