A tripyrrole peptide-Hoechst conjugate (FPH-1) has been designed which recognizes nine dA/dT base pair A/T rich dsDNA sequences at subnanomolar concentrations and complexes its targets at near diffusion controlled rates to form a fluorescent product. Spectrofluorometric titrations show the stoichiometry of the complex to be (FPH-1)(2):dsDNA. Spectrofluorometric titrations were also employed to determine the product of the equilibrium constant for complexation (K1K2) of dsDNA by two FPH-1 molecules for 35 different oligomeric duplexes. Single base pair mismatches in the FPH-1 binding site were found to cause significant decreases in K1K2 of 18- to 2300-fold. Thermal denaturation experiments provided similar results. Arguments are presented which favor the structure of the (FPH-1)(2):dsDNA minor groove complex to involve the two FPH-1 molecules in a slightly staggered, side-by-side, and antiparallel arrangement such that the bis-benzimidazole moiety of one FPH-1 molecule lies adjacent to the tripyrrole moiety of the second FPH-1 molecule.