Initiated transcription complexes were exposed to adriamycin for up to 48 h. Subsequent elongation of the transcription complex revealed the presence of a series of discrete long-lived blockage sites. The mole fraction of blocked transcripts increased linearly with reaction time, adriamycin concentration, and Fe(III) concentration. Optimal conditions for formation of the blocked transcript were 24-h reaction time, 10 microM adriamycin, and 75 microM Fe(III) ions. Nine high-intensity blocked transcripts were observed, and all correspond to transcription proceeding up to G of GpC sequences of the nontemplate strand. The presence of 75 microM Fe(III) ions enhanced the amount of transcriptional blockages by 12-15-fold. Two blocked transcripts decayed with a half-life of 0.32 and 1.9 h, and one of these exhibited 100% effective delayed termination 6 bp downstream of the original blockage site. All other blockages were unchanged after 3 h of elongation. Bidirectional transcription footprinting was used to define the physical size of the drug-induced blocking moiety as a maximum of 2 bp, and this was observed at all three GpC elements probed by RNA polymerase from both directions. The nature of the apparent covalent adducts has not yet been established but is probably a G-specific adduct deriving from a reduced form of the drug (quinone methide). Although the GpC specificity suggests an interstrand G-drug-G cross-link, these were not detected by heat denaturation and subsequent denaturing gel electrophoresis of the end-labeled promoter fragment.