Pyrrole (Py)-imidazole (Im)-containing polyamides bind in the minor groove of DNA and can recognize specific sequences through a stacked antiparallel dimer. It has been proposed that there are two different low energy ways to form the stacked dimer and that these are sensitive to the presence of a terminal formamido group: (i) a fully overlapped stacking mode in which the N-terminal heterocycles of the dimer stack on the amide groups between the two heterocycles at the C-terminal and (ii) a staggered stacking mode in which the N-terminal heterocycles are shifted by approximately one unit in the C-terminal direction (Structure 1997, 5, 1033-1046). Two different DNA sequences will be recognized by the same polyamide stacked in these two different modes. Despite the importance of polyamides as sequence specific DNA recognition agents, these stacking possibilities have not been systematically explored. As part of a program to develop agents that can recognize mismatched base pairs in DNA, a set of four polyamide trimers with and without terminal formamido groups was synthesized, and their interactions with predicted DNA recognition sequences in the two different stacking modes were evaluated. Experimental difficulties in monitoring DNA complex formation with polyamides were overcome by using surface plasmon resonance (SPIR) detection of the binding to immobilized DNA hairpin duplexes, Both equilibrium and kinetic results from SPIR show that a terminal formamido group has a pronounced effect on the affinity, sequence specificity, and rates of DNA-dimer complex formation. The formamido polyamides bind preferentially in the staggered stacking mode, while the unsubstituted analogues bind in the overlapped mode. Affinities for cognate DNA sequences increase by a factor of around 100 when a terminal formamido is added to a polyamide, and the preferred sequences recognized are also different. Both the association and the dissociation rates are slower for the formamido derivatives, but the effect is larger for the dissociation kinetics. The formamido group thus strongly affects the interaction of polyamides with DNA and changes the preferred DNA sequences that are recognized by a specific polyamide stacked dimer.