The 4-methoxypyrrolic natural products contain a common 4-methoxy-2,2'-bipyrrole chromophore and exhibit promising anticancer, antimicrobial, and immunosuppressive activities. Herein, the ability of two representative members, tambjamine E (1) and prodigiosin (2), to bind calf thymus DNA (CT-DNA), polyd[G-C](2), and polyd[A-T](2) has been characterized using absorption and fluorescence spectroscopy. Scatchard plots showed that 1 occupies a site size (n) of ca. three base pairs and possesses affinity constants (K) ranging from 1 to 0.1 x 10(5) M(-)(1). Prodigiosin (2) binds DNA by mixed modes, as isobestic points were not evident in titration experiments. The neutral aldehyde precursor 4 was found to possess no measurable DNA binding affinity, indicating that the enamine structure of 1 and the pyrromethene of 2 are essential elements for DNA binding affinity. The enamine of 1 was found to undergo hydrolysis to 4 with a half-life (t(1/2)) of 14.5 h at pH 7.4 and 37.5 degrees C. For the B-ring nitrogen atom of 1, a pK(a) value of 10.06 was also established. From fluorescence spectroscopy it was found that 1, 2, and 4 possess weak emission spectra in water that is increased in nonaqueous solvents. For 1 and 2, DNA binding also increased the emission yield. Energy-transfer measurements suggested an intercalative binding mode, with preference for AT sites. The ability of distamycin to displace 1 and 2 from the helix also suggested that they intercalate from the minor-groove. This specificity differs from other unfused aromatic cations that bind by a minor-groove mode at AT sequences and intercalate at GC sites. Reasons for the specificity displayed by 1 and 2, as well as the implications of our findings to their biological properties are discussed.