The most common chromosomal translocation in cancer, t(14;18), occurs at the bcl-2 major breakpoint region (Mbr) in follicular lymphomas. The 150-bp bcl-2 Mbr, which contains three breakage hotspots (peaks), has a single-stranded character and, hence, a non-B DNA conformation both in vivo and in vitro. Here, we use gel assays and electron microscopy to show that a triplex-specific antibody binds to the bcl-2 Mbr in vitro. Bisulfite reactivity shows that the non-B DNA structure is favored by, but not dependent upon, supercoiling and suggests a possible triplex conformation at one portion of the Mbr (peak I). We have used circular dichroism to test whether the predicted third strand of that suggested structure can indeed form a triplex with the duplex at peak I, and it does so with 1:1 stoichiometry. Using an intracellular minichromosomal assay, we show that the non-B DNA structure formation is critical for the breakage at the bcl-2 Mbr, because a 3-bp mutation that disrupts the putative peak I triplex also markedly reduces the recombination of the Mbr. A three-dimensional model of such a triplex is consistent with bond length, bond angle, and energetic restrictions (stacking and hydrogen bonding). We infer that an imperfect purine/purine/pyrimidine (R.R.Y) triplex likely forms at the bcl-2 Mbr in vitro, and in vivo recombination data favor this as the major DNA conformation in vivo as well.