Psorospermin is a DNA-reactive natural product isolated from the roots of the tropical African plant Psorospermum febrifugum that shows significant promise as an antileukemic agent. Incubation of this antineoplastic agent with DNA results in the production of sequence selective abasic sites on the DNA. Using high-field NMR and gel electrophoresis, the mechanism of covalent modification of DNA and the mode of interaction with DNA are determined. Psorospermin intercalates the DNA molecule, positioning the tricyclic xanthone chromophore in an orientation parallel to the adjacent base pairs. This places the epoxide in the major groove, resulting in site-directed electrophilic addition of the epoxide to N7 of guanine located to the 3’ side of the site of DNA intercalation. It is proposed in this study that the subsequent depurination of the psorospermin-N7-guanine adduct is the source of the previously observed in vivo formation of abasic sites on the DNA. Significantly, although the chemical structure and mechanism of covalent modification of DNA are very similar to those of the pluramycin class of agents, there are distinct differences in the relative reactivities and sequence selectivity between psorospermin and pluramycin-like compounds that may give rise to observed variances in biological activity. Specifically, while psorospermin is much less reactive than most of the pluramycins, it shows unique selectivity for 5’GG* sequences (the asterisk designates the site of covalent modification), which is the least reactive site for all of the pluramycins investigated thus far. On the basis of the solution MMR structure of the psorospermin-DNA covalent adduct, the underlying structural differences that give rise to this lower reactivity and different sequence specificities are proposed.