We have examined the capacity of oligonucleotide-directed triple helices to block the progress of primer extension by DNA polymerase. Occupancy of the major groove of a double-helical DNA substrate obstructed Klenow fragment progress at sites that map near the proximal boundary between duplex and triplex. Among a family of related third-strand oligonucleotides that all stably occupied the target duplex in the absence of polymerase, those forming longer triplexes were more effective polymerase inhibitors than shorter complexes. Kinetic analysis revealed that the triple-helical complex provided an effective blockade for times of at least 20 min. These observations provide the basis for considering and further dissecting repair DNA polymerase function and mechanism by using various defined local three-stranded DNA structures as probes.