The pluramycin antitumor antibiotics, which include the altromycins, pluramycin, hedamycin, and rubiflavin, are a group of highly evolved DNA-reactive compounds that have structural features reminiscent of both nogalamycin and the aflatoxins. As such, they are characterized as "threading intercalators" with the added ability to alkylate N7 of guanine (see preceding article in this issue). In this article we have demonstrated that different members of this group of antibiotics have sequence specificities that differ for the base pair to the 5’ side of the alkylated guanine and also have a range of reactivities with susceptible sequences. Subsequent experiments were designed to determine the molecular origin for both these observed contrasting sequence specificities and covalent reactivities. First, neopluramycin, an analog of pluramycin that lacks the epoxide, and thus is unable to covalently modify DNA, but is in other respects structurally similar, exhibits no discernible sequence selectivity.