Many paramyxoviruses express small basic C proteins, from an alternate, overlapping open reading frame of the P gene mRNA, which were previously found to inhibit mRNA synthesis. During recent experiments in which infectious Sendai virus (SeV) was recovered from cDNA via the initial expression of the viral N, P, and L genes from plasmids, the abrogation of C protein expression from the plasmid P gene was found to be necessary for virus recovery. We have investigated the effect of C coexpression on the amplification of an internally deleted defective interfering (DI) genome directly in the transfected cell, for which, in contrast to virus recovery experiments, genome amplification is independent of mRNA synthesis carried out by the SeV polymerase. We find that C protein coexpression also strongly inhibits the amplification of this DI genome but has little or no effect on that of a copy-back DI genome (DI-H4). We have also characterized the C protein from a mutant SeV and found that (i) it had lost most of its inhibitory activity on internally deleted DI genome amplification and (ii) its coexpression no longer prevented the recovery of SeV from DNA. However, consistent with the insensitivity of copy-back DI genomes to C protein inhibition, C coexpression did not prevent the recovery of copy-back nondefective viruses from DNA. The inhibitory effects of C coexpression thus appear to be promoter specific.