Single-stranded DNA of the lacUV-5 promoter formed at the active site of Escherichia coli RNA polymerase during transcription is specifically cleaved by the redox active tetrahedral cuprous chelates of 1,10-phenanthroline and its derivatives. The cleavage sites are observed in the open, initiating, and elongating complexes. Redox-inert, tetrahedral cuprous chelates of neocuproine (2,9-dimethyl-1,10- phenanthroline) and its 5-phenyl and 4-phenyl derivatives protect the template strand of DNA from scission within these steady state intermediates and inhibit transcription. Although these cuprous chelates of neocuproine bind at multiple sites within three distinct enzyme intermediates, the highest affinity site is within the elongation complex. The I50 of 5 microM for the 2:1 5-phenylneocuproine cuprous complex ((5 phi NC)2Cu+) in runoff transcription therefore primarily reflects its intermediate. The neocuproine cuprous chelates are novel transcription inhibitors because they bind to single-stranded DNA sites generated during the course of catalysis by RNA polymerase.