A synchronized in vitro transcription assay has been used to probe the sequence specificity of alkylation of DNA by nitrogen mustard. Transcriptional blockages were detected with use of a 497-base-pair PvuII/SalI restriction fragment of a modified pBR322 vector when initiation of transcription was commenced after the DNA had been alkylated but not if the initiated transcription complex was subjected to alkylation before the elongation phase. The intensity of transcriptional blockages increased with alkylation time and was maximal after 1.5 h at a mustard concentration of 200 microM. There was also evidence of alkylation of the promoter region with increasing mustard concentration. The transcriptional blockage pattern changed at some sites as elongation time was increased and three types of blockages were observed-partial transcription (one or two nucleotides) past an initial blockage site, delayed but normal transcription past some sites, and complete termination at most sites. Eight of the nine blockage sites detected were at G or GG sequences on the template strand, with an apparent specificity for 5'-CTGT sequences of the template strand. Seven of the nine sites were capable of inter- or intrastrand cross-links, including three possible G-G interstrand cross-links spanning an intervening base-pair. In the 103-bp segment probed by this procedure, transcriptional blockages were detected (with one exception) only at sites corresponding to G on the template strand where inter- or intrastrand cross-linking was possible but not for similar sequences on the non-template strand.