DNA containing the lac UV5 promoter was alkylated using bifunctional sulfur and nitrogen mustards and a monofunctional sulfur mustard. The alkylation sites were mapped using Taq polymerase, and the effect of alkylation on the formation of the DNA-RNA polymerase complex was determined using gel retardation. Alkylation was observed at all G residues in the template strand. Exposure of the alkylated DNA to Escherichia coli RNA polymerase resulted in the formation of a DNA-enzyme complex that was more stable, prior to initiation, than the complex formed with nonalkylated DNA. The DNA-RNA polymerase complex formed with the alkylated DNA also demonstrated decreased ability to progress along the full length of the DNA template. These observations show that, in addition to inducing transcriptional blockages, mustards also influence the interaction between RNA polymerase and its promoter. The ability to interfere with protein-DNA interactions may contribute significantly to the effects of these compounds in eukaryotic systems with their complex array of transcription factors.