Preferential psoralen photobinding sites have been mapped in vitro on restriction fragments spanning the SV40 origin region and surrounding sequences by a new fine structure analysis technique. Purified DNA fragments were photoreacted with 3H-5-methylisopsoralen (3H-5-MIP), a psoralen derivative which forms only monoadducts. Fragments were then end-labeled and digested with lambda exonuclease, a 5' processive enzyme which we have determined pauses at 5-MIP monoadducts. When photobinding sites were mapped on denaturing sequencing gels, it was observed that 5-MIP binds preferentially to 5'-TA sites, and to a lesser degree to 5'-AT sites. Utilizing this approach, we have identified a psoralen hypersensitive region in which the binding sites were much stronger than those in the surrounding sequences. This region extends from 150 base pairs (bp) to the late side of the enhancers to the early enhancer/promoter boundary. We suggest that this region contains a sequence directed structural alteration of the DNA helix which can be detected by the psoralen mapping approach described.