Abstract 1. 1. Five different RNA components sedimenting at 45 S, 30–34 S, 22 S, 15 S and 3 S can be resolved by a glycerol-density gradient centrifugation for the Qβ RNA polymerase reaction in vitro. 2. 2. Both the 45-S and 34-S components were shown to be enzyme-RNA complexes, which are capable of synthesizing RNA only by the addition of ribonucleoside triphosphate substrates. Upon deproteinization 30-S and 15–18-S RNA species were formed from the 45-S complex and only 30-S RNA species from the 34-S complex. 3. 3. The 22-S and 15-S components appeared late in the reaction and neither contained enzyme activity nor could act as template. By these sedimentation properties and their ribonuclease resistance, they resemble forms previously termed “replicative intermediate” and “replicative form”. 4. 4. When pulse-labeled at the beginning of reaction, the 45-S and 3-S components appeared first, followed by a decrease in the 3-S and an appearance of the 30–34-S, 22-S and 15-S components. 5. It is therefore concluded that there exist two types of enzyme-RNA complex, the 45 S and 34 S, during replication and they may function as an active replicating site of Qβ RNA in vitro.