An in vitro transcription assay was used to determine the sequence specificity of binding of mitoxantrone to a 497 bp fragment of DNA containing the lac UV5 promoter. Transcriptional blockages of the E. coli RNA polymerase were observed dominantly prior to 5'-CpA sequences (64% occurrence), and to a lesser extent 5'-CpG sequences (29%). Overall, 93% of all blockage sites were prior to pyrimidine (3'-5') purine sequences. An effect of flanking sequences was evident since the blockage sites contained an A/T base pair 5' prior to the consensus CpA and CpG intercalation sites. The consensus sequences for the preferred mitoxantrone intercalation sites are therefore 5'-(A/T)CA and 5'-(A/T)CG. The location of transcriptional blockages one base pair prior to the intercalation site is consistent with the fact that the mitoxantrone side chains lie in the major groove.