N,N'-Bis(2-chloroethyl)-N-nitrosourea (BCNU) and its derivatives are chemotherapeutic DNA-damaging agents that generate a variety of monoadducts, intrastrand cross-links, and interstrand cross-links. The cytotoxic potential of the compounds has been linked to their ability to form DNA interstrand cross-links, which presumably inhibit subsequent DNA replication. To address the possibility that BCNU-induced lesions may also influence other DNA-directed actions such as transcription, and to identify the DNA lesions involved, a synthetic DNA template containing phage RNA polymerase promoters at both ends was incubated with BCNU and, after drug removal, transcribed in vitro. For comparison, similar studies were carried out with cis-diammine-dichloroplatinum(II) and trans-diamminedichloroplatinum(II), which are known to induce defined transcription-terminating lesions. The results suggest that BCNU, like platinum compounds, can induce lesions resulting in termination of transcription in vitro, although the predominant transcription-terminating lesions, unlike those produced by cis-diamminedichloroplatinum(II), most likely represent interstrand DNA cross-links.