The generation of defective-interfering (DI) particles by rubella virus during serial undiluted passage and persistent infection in Vero cells was studied. A series of 24 serial undiluted passages was initiated with plaque-purified virus. The virus titer remained relatively constant through the first nine passages, after which it declined, reaching a low level of 20-fold less than the originating stock by passage 15. In subsequent passages, the titer cycled. Intracellular DI RNAs were first detectable at passage 4, at which time DI RNAs of 7500 and 1400 nucleotides in length were observable. Thus, the rate of which DI RNAs were generated by rubella virus during serial undiluted passage was similar to the rate of DI generation by other enveloped RNA viruses during serial undiluted passage. The longer rubella DI RNA was present in all passages subsequent to passage 4, while the 1400-nucleotide DI RNA was replaced by a DI RNA of 800 nucleotides in length by passage 15. Subsequent to passage 7, the relative amount of genomic RNA declined dramatically and the DI RNAs became the predominant intracellular virus-specific RNA species. Negative-polarity RNA species corresponding to the 7500- and 800-nucleotide DI RNA species were identified. The 7500- and 1400-nucleotide DI RNA species were encapsidated into virus particles while the presence of the 800-nucleotide DI RNA species in virus particles could not be detected. Interestingly, the rubella virus subgenomic RNA was present in virus particles in preparations containing DI RNAs. A persistent infection was initiated by subculturing the surviving cells from a high multiplicity of infection with plaque-purified virus. Intracellular DI RNAs were first detectable at Day 19 after initiation of persistence and became significant by Day 26. The amount of genomic RNA began to decrease at Day 47 and was undetectable after Day 68. Through Day 54, there were several DI RNA species present, but at later times, one of these species became predominant. Thus, DI particles were generated during persistent infection, but their presence was not necessary for initiation of persistence.