An in vitro transcription assay has been used to define twelve high occupancy transcriptional blockage sites in 260 bp of heterogenous DNA on three promoter-containing DNA fragments. Transcription proceeded to immediately upstream of CpA sequences in nine of these sites, and this defines the most preferred intercalation site as CpA. In almost all cases, this sequence was flanked by T on the 5' end. The consensus sequence for the highest affinity Adriamycin site is therefore 5'-TCA, with some evidence for preference of AT base pairs flanking both ends of this trinucleotide [i.e., (t)TCA(a.t)(a.t)]. In contrast, lower occupancy CpA sites were flanked on the 5' end by a GC base pair, with a preference for up to three GC base pairs on the 3' end. The low affinity consensus sequence is therefore (G.C)CA(g.c)(g.c)(g.c).