i-Motif-forming sequences are present in or near the regulatory regions of >40% of all genes, including known oncogenes. We report here the results of a biophysical characterization and computational study of an ensemble of intramolecular i-motifs that model the polypyrimidine sequence in the human c-MYC P1 promoter. Circular dichroism results demonstrate that the mutant sequence (5'-CTT TCC TAC CCTCCC TAC CCT AA-3') can adopt multiple i-motif-like, classical i-motif, and single-stranded structures as a function of pH. The classical i-motif structures are predominant in the pH range 4.2-5.2. The i-motif-like and single-stranded structures are the most significant species in solution at pH higher and lower, respectively, than that range. Differential scanning calorimetry results demonstrate an equilibrium mixture of at least three i-motif folded conformations with Tm values of 38.1, 46.6, and 49.5 degrees C at pH 5.0. The proposed ensemble of three folded conformations includes the three lowest-energy conformations obtained by computational modeling and two folded conformers that were proposed in a previous NMR study. The NMR study did not report the most stable conformer found in this study.