John A. Thomas, 1 Robert N. Buchsbaum, Andrzej Zimniak, and Efraim Racker* abstract: The uncharged, colorless molecule fluorescein diacetate diffuses into Ehrlich ascites tumor cells at neutral pH, where intracellular esterases release the chromophore fluorescein. The negatively charged dye is retained by the cell, permitting the intracellular pH to be estimated from the shape of the pH-dependent absorption spectrum. The diacetate derivative of 6-carboxyfluorescein may be used similarly and has the additional advantage of a slower rate of leakage out of the cell but requires incubation at pH 6.2 to facilitate initial entry into the cell. After removal of external dye by cen-trifugation, 80-92% of the remaining dye is unresponsive to external pH changes. Calibration of the intracellular fluorescein spectra is obtained by equilibration of the internal and external pH with nigericin in K+ buffers. Results of intracellular pH measurements by this method are in good agreement with those obtained by measuring the distribution