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Pancreatic β-cell imaging would be useful in monitoring the progression of and therapies for diabetes. The purpose of this study was to develop and evaluate quantitative β-cell MRI using manganese (Mn2+) labeling of β cells, T1 mapping, and a two-site water exchange model. Normal, pharmacologically-treated, and severely diabetic mice underwent injection of MnCl2. Pancreatic water proton T1 relaxation was measured using Look-Locker MRI, and two-site water exchange analysis was used to estimate model parameters including the intracellular water proton relaxation rate constant (R1ic) and the intracellular fraction as indicators of β-cell function and mass, respectively. Logarithmic plots of T1 relaxation revealed two distinct proton pools relaxing with different T1s, and the two-site water exchange model fit the measured T1 relaxation data better than a monoexponential model. The intracellular R1ic time course revealed the kinetics of β-cell Mn2+ labeling. Pharmacological treatments with nifedipine, tolbutamide, and diazoxide altered R1ic, indicating that beta cell function was a determinant of Mn2+ uptake. Intracellular fraction was significantly higher in mice with normal β cell mass than in diabetic mice (14.9% vs. 14.4%,P< 0.05). Two-site water exchange analysis of T1 relaxation of the Mn2+-enhanced pancreas is a promising method for quantifying β cell volume fraction and function.