Adriamycin and mitomycin C were reduced by xanthine oxidase/NADH in the presence of a DNA template comprising a stable initiated ternary transcription complex derived from the lac UV5 promoter. Subsequent elongation of the transcription complex treated with mitomycin C revealed high levels of terminated transcripts one nucleotide prior to G residues on the coding strand (i.e. at X of XpC sequences of the non-coding strand). Lower levels of termination occurred with adriamycin, and these were also one nucleotide prior to G residues of the coding strand, but with greater sequence specificity since they were observed mainly at G of GpC sequences of the non-coding strand. The same sites were also observed with adriamycin in the absence of reducing conditions and the level of termination at these sites was enhanced up to 10-fold by Fe2+ and Fe3+, but not by Cu2+, Zn2+, Co2+ or Ni2+. These results suggest that an iron-adriamycin complex with DNA is highly sequence-specific and results in adducts, similar to those of mitomycin C, which can terminate the transcription process. Such a mechanism offers new insights into the possible mode of action of anthracyclines.