G center dot C Hoogsteen base pairs can form transiently in duplex DNA and play important roles in DNA recognition, replication, and repair. GC Hoogsteen base pairs are thought to be stabilized by protonation of cytosine N3, which affords a second key hydrogen bond, but experimental evidence for this is sparse because the proton cannot be directly visualized by X-ray crystallography and nuclear magnetic resonance spectroscopy. Here, we combine NMR and constant pH molecular dynamics simulations to directly investigate the pK(a) of cytosine N3 in a chemically trapped N1-methyl-G center dot C Hoogsteen base pair within duplex DNA. Analysis of NMR chemical shift perturbations and NOESY data as a function of pH revealed that cytosine deprotonation is coupled to a syn-to-anti transition in N1-methyl-G, which results in a distorted Watson-Crick geometry at pH >9. A four-state analysis of the pH titration profiles yields a lower bound pK(a) estimate of 7.2 +/- 0.1 for the G center dot C Hoogsteen base pair, which is in good agreement with the pK(a) value (7.1 +/- 0.1) calculated independently using constant pH MD simulations. Based on these results and pH-dependent NMR relaxation dispersion measurements, we estimate that under physiological pH (pH 7-8), G center dot C Hoogsteen base pairs in naked DNA have a population of 0.02-0.002%, as compared to 0.4% for A center dot T Hoogsteen base pairs, and likely exist primarily as protonated species.