An in vitro transcription assay has been developed to define the exact location of DNA binding ligands. The method employs two counterdirected Escherichia coli promoters separated by approximately 100 bp. Selective transcription from each promoter yields transcripts up to each ligand site. Nonsaturating levels of ligands result in fractional occupancy of ligand at each site, and hence a range of RNA transcript lengths. The bidirectional promoter system results in a transcription footprint which was derived from transcription from both promoters up to the 5' side of each occupied ligand site and defines the sequence specificity and binding site size of the DNA-bound ligand. The transcriptional footprint is precise to +/- 1 bp from the 5' and 3' ends of each binding site. Multiple ligand sites can be ranked in terms of relative fractional occupancy at each site, and the ranking is comparable from either transcription direction. The method was compared to classical DNase I footprinting with a series of DNA binding drugs [actinomycin D, echinomycin, bis(thiadaunomycin), mithramycin, nogalamycin, and an acridine-tripyrrole]. In all cases, specific binding sites were resolved more clearly by transcription footprinting than by DNase I footprinting. Because of the nature of the transcription assay, all occupied ligand sites were detected by this method, in contrast to DNase I footprinting where many sequences are not probed, and where ligand sites are often not accurately defined.