Ecteinascidin-743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from the tunicate Ecteinascidia turbinata currently under phase II clinical trials for its potent anticancer activity. ET-743 binds DNA in the minor groove and forms covalent adducts with some sequence specificity. It selectively inhibits in vitro binding of the CCAAT box factor NF-Y. In this study, we assayed ET-743 function in vivo on the HSP70 promoter. On heat induction, the drug blocks transcription rapidly at pharmacological concentrations and in a CCAAT-dependent manner, whereas the activity of the CCAAT-less simian virus 40 promoter is not affected. The effect is exerted at the mRNA level. The distamycin-like alkylating tallimustine is inactive in these assays. Binding of NF-Y and of the heat-shock factor is normal in ET-743-treated cells. Run-on analysis of several endogenous genes further proves that the drug has rapid, profound, and selective negative effects on transcription. Thus, this marine-derived compound is a promoter-specific, transcription-interfering agent.