The reaction of cyanomorpholinoadriamycin (CMA) with DNA results in the formation of sequence-specific complexes with DNA. These complexes were revealed as blocked transcripts in an in vitro transcription assay--of 14 high-intensity blockages detected in the 120 bp probed in this assay, 12 were prior to GpG or CpC sequences. Slow read-through past the first few sites exhibited first-order kinetics, with half-lives of 25-200 min. Bidirectional transcription footprinting revealed nine high-intensity sites, eight of which were defined by a GpG element (nontemplate strand). Reaction of CMA with single-strand DNA, followed by a primer-extension assay, revealed four major blockages all of which were at GpG sites on the initial single-strand DNA. From a combination of these three experimental approaches, it appears that CMA yields dominantly intrastrand cross-links between adjacent guanine residues. Since CMA is also known to form interstrand cross-links, these appear to occur at GpC sequences but are minor in comparison to the extent of formation of intrastrand cross-links.