Changes in intracorneal and storage-medium pH values of organ-culture-stored cat corneas were monitored over a 4-week period. The intracorneal pH was determined using the phosphorus-31 magnetic resonance spectroscopy (31P MRS) chemical shift of inorganic orthophosphate in conjunction with a standard pH titration curve. We incubated 32 adult cat corneas using two similar standard organ-culture methods, one with chondroitin sulfate (method 1) and the other without (method 2). Time-course data at 0, 1, 3 and 4 weeks of storage were used to calculate the rate of pH change. The intracorneal pH was not changed significantly for either organ-culture method; however, the storage-medium pH rate of change declined significantly for both methods (method 1, 0.15 pH units/week; method 2, 0.12 pH units/week). The difference between intracorneal and storage-medium pH values over time increased at a rate of 0.12 and 0.11 pH units/week for method 1 and method 2, respectively. The declining storage-medium pH in conjunction with the maintenance of intracorneal pH contributes to an increased metabolic demand on the cornea.