Imidazole and pyrrole-containing polyamides belong to an important class of compounds that can be designed to target specific DNA sequences, and they are potentially useful in applications of controlling gene expression. The extent of polyamide curvature is an important consideration when studying the ability of such compounds to bind in the minor groove of DNA. The current study investigates the importance of curvature using polyamides of the form f-Im-Phenyl-Im, in which the imidazole heterocycles are placed in ortho-, meta-, and para-configurations of the phenyl moiety. The synthesis and biophysical evaluation of each compound binding to its cognate DNA sequence (5-ACGCGT-3) and a negative control sequence (5-AAATTT-3) is reported, along with their comparison to the parent binder, f-Im-Py-Im (3). ACGCGT is a medicinally significant sequence present in the MluI cell-cycle box (MCB) transcriptional element found in the promoter of a gene associated with cell division. The results demonstrated that the para-derivative has the greatest affinity for its cognate sequence, as indicated via thermal denaturation, CD, ITC, SPR analyses, and DNase I footprinting. ITC studies showed that binding of the para-isomer (2c) to ACGCGT was significantly more exothermic than binding to AAATTT. In contrast, no heat change was observed for binding of the meta- (2b) and ortho- (2a) isomers to both DNAs, due to low binding affinities. This is consistent with results from SPR studies, which indicate that the para-derivative binds in a 2:1 fashion to ACGCGT and binds weakly to ACCGGT (K = 1.8 x 106 and 4.0 x 104 M-1, respectively). Interestingly, it binds in a 1:1 fashion to AAATTT (K = 5.4 x 105 M-1). The metacompound does not bind to any sequence. The para-derivative also was the only compound to show an induced peak via CD at 330 nm, indicative of minor groove binding, and produced a ΔTm value of 5.8 °C. Molecular modeling experiments have been performed to determine the shape differences between the three compounds, and the results indicate that the para-derivative 2c has a closest curvature to previously synthesized polyamides. DNase I footprinting studies confirmed earlier observations that only the para-derivative 2c produced a footprint with ACGCGT (1 μM) and no significant footprint was observed at any sites examined for meta-2b and ortho-2a analogs up to 40 μM. The results of these studies suggest that the shape of the ortho- and meta- derivatives is too curved to match the curvature of the DNA minor groove to facilitate binding. The para-derivative gives the highest binding affinity in the series and the results illustrate that 4- aminobenzamide is a reasonable substitute for 4-aminopyrrole-2-carboxylate.