The L double-stranded (ds) RNA component of Saccharomyces cerevisiae may contain up to three dsRNA species, each with a distinct sequence but with identical molecular weights. These dsRNAs have been separated from each other by denaturation and polyacrylamide gel electrophoresis. The 3' terminal sequences of the major species, LA dsRNA, were determined. Secondary structural analysis supported the presence of two stem and loop structures at the 3' terminus of the LA positive strand. In strain T132B NK-3, both the LA and LC species are virion encapsidated. Two distinct classes of virions were purified from this strain, each with a different RNA polymerase activity and with distinct protein components. The heavy virions harbored LA dsRNA, whereas the LC dsRNA species co purified with the light virion peak. Thus, LA and LC dsRNAs, when present in the same cell, may be separately encapsidated.