Autosomal dominant polycystic kidney disease (ADPKD) affects over 500,000 Americans; one-half of which progress to end-stage renal disease by 50 years of age. Eighty-five percent of these patients have mutations in the PKD1 gene. Recent investigations have demonstrated that cyst formation may result from loss of function at this genetic locus secondary to loss of heterozygosity. This would require a very high mutation rate to explain the large number of cysts present in affected kidneys; therefore, we hypothesized that some inherent features of the PKD1 gene predispose it to mutation. Intron 21 of this gene contains the largest polypurine polypyrimidine tract (2.5 kb) identified in the human genome. Polypurine polypyrimidine mirror repeats are known to have anomalous helical characteristics, form intramolecular triplexes and predispose to mutagenesis in vivo. Our studies reveal that this fragment from intron 21 has anomalously increased electrophoretic mobility implying a different helical nature. We used circular dichroism to study the properties of the helix and its ability to form triplex DNA. Supercoiled plasmid with the cloned intron 21 or the isolated fragment was placed in 10 mM sodium phosphate in a range of pH conditions from pH 5.5 to 7.5, and then analyzed by circular dichroism. Preliminary results support the notion that at pH 7.5, the isolated 2.5 kb PKD1 segment may have A-form helical characteristics. A significant dampening after 260 nm was seen only at pH 5.5, indicating a change in the helical nature of the DNA with protonation compatible with triplex formation. Using two-dimensional gel electrophoresis, we further evaluated the conformational changes adopted by intron 21 in a recombinant plasmid. We observed a sharp structural transition by the insert under conditions of negative supercoiling and pH <7. The superhelical density at which a transition was observed was linearly related to pH confirming that the structure under study was protonated. These results are most consistent with the formation of an intramolecular triplex structure. This structural characteristic is strongly implicated in DNA recombination and mutagenesis, and may contribute to both somatic and germline mutagenesis in the PKD1 gene.