Synthesis of the vesicular stomatitis virus nucleocapsid (N) protein is required for viral RNA replication. The observation that the N protein forms a rapidly sedimenting species in the absence of other viral proteins and the description of complexes of N protein with NS protein led to the proposal that NS protein binds to N protein to prevent it from self-associating. We tested this model by analysing the physical properties of N protein synthesized alone in an in vitro replication system as compared to N protein synthesized in the presence of the NS protein. These findings were correlated with the ability of the N protein, synthesized under both conditions, to support replication. N protein synthesized at low concentrations in the absence of other viral proteins sedimented at 4S on glycerol gradients and was capable of supporting RNA replication. In contrast, synthesis of increasing concentrations of N protein resulted in formation of a rapidly sedimenting species of N protein which had the physical properties of a protein-protein aggregate and which failed to support RNA replication. Co-synthesis of the NS protein with N protein both prevented the concentration-dependent aggregation of N and restored the ability of high concentrations of N protein to support RNA replication.