Black beetle virions contain two RNAs. The smaller one, RNA 2, has previously been shown to be a messenger for viral coat protein. It is shown here, by infecting sensitized Drosophila cells with the individually purified RNAs, that the larger one, RNA 1, carries the viral gene(s) required for RNA polymerase functions. RNA 2 was dispensible for synthesis of viral RNA 1 and subgenomic RNA 3 but was essential for synthesis of RNA 2 and virions. Cells infected with RNA 1 alone produced RNA 3 in proportions 10- to 20-fold greater than cells infected with virions. This overproduction of RNA 3 decreased with increasing proportions of RNA 2 in the infecting RNA 1. We conclude that RNA 1 is the previously unidentified progenitor of subgenomic RNA 3, whereas RNA 2 regulates the amount of RNA 3 produced in the infected cell.