The intracellular pH (pH<sub>i</sub>) of basilar artery rings was determined with 2’,7’-bis-(carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) by measuring the ratio of emitted (540 nm) fluorescence intensities (FI) at excitation wavelengths of 500 and 440 nm. There was a dye loss from the rings in 90 min (39.3 ± 3.6%; p < 0.001). We found that the ratio of fluorescence intensities does not adequately correct for dye loss; hence, we derived a method to correct for dye loss during pHi determinations. Calibration curves of the ratio versus pH, were constructed for the artery rings. The slope and intercept of the calibration curves depended on FI<sub>440</sub>. Linear regression lines for the slope and intercept versus FI440 were: slope = 0.00343 × FI<sub>440</sub> + 0.32484 (r – 0.96; p < 0.0001) and intercept = –0.02048 × FI<sub>440</sub> – 0.80123 (r = –0.97; p < 0.0001). In solutions with different pH and different concentrations of free BCECF, the slope of the ratio versus pH of the solution was steeper at high concentrations of BCECF. Thus, pH was calculated from a calibration curve in which the slope and intercept were determined from FI440 with the above formula. The corrected pH<sub>i</sub> was 7.37 ± 0.05 (n = 25) at pH<sub>o</sub> 7.4 and 37 °C.