RNA from purified bovine enterovirus (serotype VG-5-27) was fractionated by sedimentation on sucrose gradients, gel filtration through Sepharose 2B and electrophoresis in acrylamide-agarose gels. Virus RNA was single stranded with a molecular weight of approximately 2.8 × 106. The RNA obtained from virus infected cells was separated into replicative intermediate and single-stranded molecules by gel filtration through Sepharose 2B. Electrophoresis of the replicative intermediate fraction on acrylamide-agarose gels gave two peaks of radioactivity, whereas after ribonuclease treatment only a single peak was present. Electrophoresis of the single-stranded molecules on acrylamide gels showed that a portion of these had slower mobilities than RNA from purified virus. This single-stranded fraction contained molecules with molecular weights in the range 5.6 to 2.8 × 106. Continuous labelling experiments showed that this size range was a function of time after infection. The existence of single strands of RNA longer than RNA found in purified virus was interpreted as supporting a cyclic model for RNA replication.