In an attempt to determine the mechanism (or mechanisms) by which vesicular stomatitis virus (VSV) kills cells, products of VSV transcription were tested in a cell-free system for their capacity to inhibit transcription of SV40 DNA and plasmids containing adenovirus late promoter and adenovirus-associated RNA genes. VSV RNA transcripts and other RNAs were compared for their capacity to suppress transcription of these DNA templates by RNA polymerases and cofactors present in the HeLa-cell extract system. Relatively low concentrations of the plus-strand leader RNA made in vitro from the 3' end of the wild-type VSV genome were found to inhibit initiation of transcription catalyzed by both RNA polymerase II and RNA polymerase III. Polyadenylated VSV messengers and other natural and synthetic RNAs also caused some inhibitory effects on in vitro transcription from DNA templates, but only at extremely high concentrations. Compared with the wild-type plus-strand RNA leader, the leader RNA synthesized in vitro by defective-interfering VSV showed only limited capacity to inhibit RNA synthesis on adenovirus and SV40 DNA templates and only at concentrations at least 30 times greater than that of the wild-type leader. The existence of nucleotide sequences in wild-type leader RNA, not present in defective-interfering leader RNA, that could recognize and block promoters, polymerases or protein cofactors is discussed.