A Pichinde persitently infected BHK21/13S culture was established in which defective interfering (DI) virus continued to be synthesized after cessation of plaque-forming virus replication. This DI virus, concentrated from NaCl-polyethylene glycol treated tissue culture fluids, was shown to band over a much broader range than standard virus, in either discontinuous or continuous sucrose gradients. The polyacrylamide gel profile of the RNAs extracted from standard virus contained six components with sedimentation coefficients corresponding to 31, 28, 22, 18, 15 and 4-6S. All RNAs extracted from DI virus preparations, however, did not contain the 22 and 15S species. Furthermore, a new 20S fraction was observed in DI virus taken from cultures which had been maintained for more than 175 generations after the initial infection, whereas it was absent in DI virus synthesized prior to that time.