The three dimensional structure of the DNA oligomer d(5'GGGTACCC-3')2 cross-linked with 4'-aminomethyl-4, 5', 8-trimethylpsoralen(AMT) has been determined by two-dimensional NMR and a relaxation matrix refinement method. NMR data and structural calculation establish that the cross-linking of the psoralen in the B-DNA duplex retains Watson-Crick type hydrogen bonding throughout the duplex, although the thymine residue which is cross-linked with psoralen forms a weaker hydrogen bond. The psoralen cross-linking in DNA duplex induces significant change of the local DNA structure, but has no important effect on overall DNA helix. Our observation places an upper limit of 10 degrees on overall DNA bending by psoralen cross-linking, which is consistent with the result of HMT-octamer cross link [Spielmann, H. P., Dwyer, T. J., Sastry, S. S., Hearst, J. E., and Wemmer, D. E., (1965) Proc. Natl. Acad. Sci. USA 92, 2345-2349] and gel electrophoretic study [Haran, T. E. and Crothers, D. E. (1988) Biochemistry 27, 6967-6971].