Pancreatic duct cells secrete the bicarbonate ions found in pancreatic juice. Impairment of ductal bicarbonate secretion, as occurs in cystic fibrosis, has serious consequences for pancreatic function and for the structural integrity of the gland. As bicarbonate is a buffer ion, the accurate measurement of intracellular pH (pHi) in duct cells is an important technique for studying the mechanisms of bicarbonate transport. Commonly, pHi is measured using the fluorescent dye biscarboxyethylcarboxyfluorescein (BCECF). The purpose of this study was to develop a new technique for the accurate calibration of BCECF fluorescent signals. Our results indicate that BCECF fluorescence is not only dependent on pHi but also on the total fluorescence intensity of the detected area (which may be influenced by dye loading, dye leakage, and the shutter size on the photomultiplier). The outcome is that one calibration curve is not sufficient for accurate determination of pHi. In fact, an appropriate calibration curve must be selected for each individual experiment. Moreover, the calibration plot is only linear over a narrow range of pHi values. In conclusion, we have developed a new technique that should be applicable to all cell types for the accurate calibration of fluorescent signals from the pH-sensitive dye BCECF.