Interference with virus replication through the use of defective viral sequences is providing new insight to replication strategies and novel approaches for induced resistance. Because replication of brome mosaic virus (BMV) is potentiated by the intercistronic region of RNA-3, we examined the effect of adding various (-)sense RNAs corresponding to this region in co-transfections with wild type BMV RNAs. Progeny accumulation in barley protoplasts transfected with RNAs 1+2 was decreased by 90% in the presence of (-)RNA-3 delta HindIII, the longest (-)sense transcript tested, and by 85% when RNA-3 was also present. This trans interference was concentration dependent, and the use of deletion derivatives of (-)RNA-3 delta HindIII revealed that previously identified regulatory sequences within the intercistronic region were responsible for the observed interference. These deletion mutants were found to be of differing stabilities and several served as effective substrates for host-encoded polymerase to yield complementary (+)strands. Indeed, it is possible that the copying of viral RNA by the host polymerase serves as a hybrid arrest mechanism for discriminating against viral RNA functions. However, neither the ability of these sequences to serve as templates for host polymerase nor their (+)strand products contributed to the interference phenomenon, which may provide a new approach for engineering resistance to viral infection.