Although autosomal dominant polycystic kidney disease is transmitted in an autosomal dominant fashion, there is evidence that the pathophysiology of cystogenesis involves a second hit somatic mutation superimposed upon the inherited germline mutation within the renal tubule cells. The polypurine.polypyrimidine (Pu.Py) tract of PKD1 intron 21 may play a role in promoting somatic mutations. To better characterize this tract and to evaluate its potential to participate in mutagenesis, we investigated the thermodynamics of intramolecular triplex formation by 15 Pu.Py mirror repeat tracts from PKD1 intron 21 by 2D gel electrophoresis. We demonstrate that intramolecular triplexes form with modest superhelical tensions for all the tracts examined. Primer extension studies demonstrated significant polymerase arrest within the Pu.Py tracts in one direction of replication only. We found correlation between polymerization arrest and both the potential length of the triplex and superhelical tension of intramolecular triplex formation. The presence of a Pu.Py tract also led to a replication blockade and double-strand breakage using an SV40 in vitro replication assay with HeLa cell extracts. During DNA replication, the G-rich template of the PKD1 Pu.Py tracts may form a triplex structure with the nascent strand, thereby blocking replication and potentially leading to recombination and mutation.