PURPOSE: The aim of this study was to generate quantitative extracellular pH maps of tumors using a combination of a pH-sensitive iron chelate-based contrast agent (IBCA) and a pH-insensitive IBCA for concentration measurement, which we termed ratiometric pH magnetic resonance imaging (RpH-MRI). METHODS: The pH-sensitive IBCA of ethylenediamine-trans-cyclohexane diamine tetraacetic acid (Fe-en-tCDTA) was synthesized, along with the pH-insensitive IBCAs of trans-cyclohexane diamine tetraacetic acid (Fe-tCDTA) and diethylenetriamine-N,N,N',N″,N″-pentaacetic acid (Fe-DTPA). The pH-dependent T1 contrast effects of these chelates were compared in water and serum phantoms at 0.94 T, 3 T and 7 T. For in vivo pH mapping of tumors at 7 T, 4T1 breast cancer cells were inoculated subcutaneously into the flanks of the BALB/c mice. RpH-MRI was performed with two sequential intravenous applications: first a pH-insensitive IBCA, followed by the pH-sensitive IBCA at the same dose (0.25 or 0.5 mmol/kg) with an interval of either 30 or 60 min. Quantitative pH maps were generated by calculating T1, S, and relative maximum enhancement maps of the two injections, together with pH-dependent T1-relaxivity parameters derived from in vitro measurements of the pH-sensitive IBCA and pH-insensitive control IBCA. RESULTS: The T1 relaxivity (r1) of Fe-en-tCDTA was highly pH dependent, being approximately 2.7 times higher at pH 5.5 than at neutral pH, whereas Fe-DTPA and Fe-tCDTA showed stable r1 values between pH 5.5-7.4. In vivo, the time to maximum signal intensity (TMI) of the tumors of Fe-DTPA as control was comparable to that of Fe-en-tCDTA (2.57 ± 1.34 min vs. 2.683 ± 0.89 min, p = 0.7596, paired t test, 4 mice, 7 tumors) as well as for Fe-tCDTA as control versus Fe-en-tCDTA (3.30 ± 1.17 min vs. 3.627 ± 1.12 min, p = 0.2101, paired t test, 7 mice, 13 tumors), suggesting similar pharmacokinetics. The concentration distribution at TMI of the control chelates was assumed to be the same as that of the second injected Fe-en-tCDTA. The dynamic contrast enhanced MRI curve of the first injection of Fe-DTPA returned to baseline after 20-30 min, whereas Fe-tCDTA took 30-60 min to reach baseline. Calculated core and rim pH values were 6.512 ± 0.182 and 6.742 ± 0.121, respectively (p < 0.0001, paired t test, 11 mice, 20 tumors) with core areas showing lower chelate concentrations but higher T1 relaxivity; the mean tumor-wide pH value was 6.632 ± 0.140. CONCLUSION: Our results demonstrate the potential of high-resolution RpH-MRI based on pH-sensitive and pH-insensitive IBCAs for mapping tumor extracellular pH and concentration distribution.