An assay has been developed to detect the DNA sequence specificity of drug binding sites, and the kinetics of dissociation of drug from those sites, under conditions involving active transcription of the DNA. Specific transcriptional blockage sites were detected in the presence of actinomycin D and a bisintercalator, bis(anthracycline); the rate of RNA chain growth past the drug binding sites yields the rate of dissociation of drug from these sites. Rate constants for dissociation from the whole promoter fragment measured by the detergent sequestration method were found to be significantly faster than the rate determined for dissociation from the specific transcriptional blockage site. However, the absence of significant blockage at other drug binding sites implies much more rapid drug dissociation from those sites in the transcriptional complex. We conclude that transcriptional blockage results from a DNA sequence-dependent interaction of the drug-DNA complex with RNA polymerase; the sequences that are effective for blockage appear to be GpC for actinomycin and (CpA)3 for a bis(daunomycin) compound. Transcriptional inhibition may in general show greater sequence specificity than is exhibited by simple binding.