Methylation protection studies suggested that the NS protein component of the RNA polymerase of vesicular stomatitis virus contacts the RNA templates of defective interfering (DI) particles at the sequence 3'...GUCUAUUUUUAUUUUUGGUG...5',17 to 37 nucleotides downstream from the site of initiation of in vitro transcription. The data indicated that vesicular stomatitis virus and DI particle RNAs contain different polymerase binding sequences and that NS may function as a transcription initiator protein for template recognition at both sequences. These results are thus compatible with the hypothesis that differences in the rate of defective and nondefective viral particle replication and autointerference are due to higher-affinity binding sites for polymerase at the 3' end of DI particle RNAs. In addition, a unique DI particle (DI-LT2) RNA that contains a transcriptionally inactive vesicular stomatitis virus leader gene 72 to 118 nucleotides from its 3' end showed interactions with the viral polymerase similar to those reported previously for the 3'-terminal vesicular stomatitis virus leader gene (Keene et al., Proc. Natl. Acad. Sci, U.S.A. 78:6191--6195, 1981). The interaction of polymerase with the internal leader gene of DI-LT2 RNA suggested that the lack of leader RNA and mRNA production by this particle is not due to the inability of polymerase to bind to internal sites along the template. Instead, the initiation of transcription is more likely influenced by the position of the polymerase binding site relative to the 3' end or by requisite interactions between the catalytic polymerase component (L) and the proposed initiator protein (NS).