After the incubation of reovirus replicase reaction mixtures (containing labeled ribonucleoside triphosphates), partially double-stranded ribonucleic acid (pdsRNA) products were isolated by cellulose column chromatography followed by precipitation with 2 m NaCl. The pulse-labeled reaction product contained a significantly large amount of pdsRNA that became complete dsRNA as reaction time increased, indicating that pdsRNA was an intermediate of the replicase reaction. The newly synthesized RNA strand ((3)H-labeled) of the pdsRNA was resistant to ribonuclease digestion, suggesting that single-stranded RNA regions were part of a preexistent unlabeled RNA template. These observations, together with the electrophoretic behavior of the pdsRNA in polyacrylamide gel, are consistent with the hypothesis that dsRNA is synthesized by the elongation of a complementary RNA strand upon a preexistent template of single-stranded RNA (i.e., messenger RNA). The direction of the RNA strand elongation was determined by carrying out the replicase reaction in the presence of (3)H-cytidine triphosphate (or (3)H-uridine triphosphate) and adenine triphosphate-alpha-(32)P followed by a chase with excess unlabeled cytidine triphosphate (or uridine triphosphate). The dsRNA product was digested with T1 ribonuclease and the resulting 3'-terminal fragments were isolated by chromatography on a dihydroxyboryl derivative of cellulose. Examination of the ratio of (3)H to (32)P in these fragments indicated that RNA synthesis proceeded from the 5' to 3' terminus.