Two triple helix structures (15-mers containing only T.A-T triplets or containing mixed T.A-T and C.G-C triplets) have been studied by uranyl mediated DNA photocleavage to probe the accessibility of the phosphates of the DNA backbone. Whereas the phosphates of the pyrimidine strand are at least as accessible as in double stranded DNA, in the phosphates of the purine strand are partly shielded and more so at the 5'-end of the strand. With the homo A/T target increased cleavage is observed towards the 3'-end on the pyrimidine strand. These results show that the third strand is asymmetrically positioned along the groove with the tightest triple strand double strand interactions at the 5'-end of the third strand. The results also indicate that homo-A versus mixed A/G 'Hoogsteen-triple helices' have different structures.