The interactions of several pyrrolo[2, 1-c][1,4]benzodiazepine (PBD) antitumor antibiotics with linearized plasmid pGEM-2-N-ras DNA have been analyzed by quantitative in vitro transcription (QIVT) and in vitro transcription footprinting (IVTF) methods. A concentration-dependent inhibitory effect of the PBDs on transcription is observed using both techniques. The rank order for overall inhibition of transcription by the QIVT method is found to be: sibiromycin > tomaymycin > anthramycin > DC-81 > neothramycin, whereas the IVTF experiments show a different ranking: sibiromycin > anthramycin > neothramycin > tomaymycin. In addition, stimulation of transcription was observed at low PBD concentrations in both the QIVT and IVTF experiments. These results demonstrate unequivocally that the formation of PBD-DNA adducts at AGA-5' base sequences on the transcribed strand results in transcription blockage for all PBDs examined. Furthermore, the sequence of flanking base pairs appears to influence the degree of blocking, with the sequences ACAGAAA-5', AAAGATG-5', AGAGATA-5', and CAAGAAC-5' providing the most pronounced blocks for all PBDs studied in this system. Neothramycin and tomaymycin cause additional blocks at some GGA-5' and TGA-5' sequences. Parallel MPE-Fe(II) footprinting studies have revealed PBD binding sites on both the transcribing and nontranscribing strands, although all transcription blocks determined from the IVTF assays are due to drug bound on the transcribing DNA template strand.