The green fluorescent protein of Aequorea victoria (GFP) is a natural peptide chromophore without substrate or cofactor requirements for fluorescence. In vitro, a recombinant F64L/S65T GFP mutant (GFPmut1) exhibited pH sensitive fluorescence within the physiologic range. When heterologously expressed in BS-C-1 cells or rabbit proximal tubule cells, uniform cytosolic and nuclear fluorescence was observed. Cytosolic fluorescence constituted over 80% of the total. Excitation scanning of transfected cells revealed two GFPmut1-specific regions that were pH-sensitive over the physiologic range, and each region exhibited a unique pH "bias" in fluorescence emission. Excitation at or near the expected maximum of 488 nm (region II) uniformly resulted in fluorescence that was preferentially altered at acidic pH. In contrast, a novel "wild-type" excitation peak at 400 nm (region I) resulted in alkaline-biased fluorescence similar to that described for the wild-type chromophore in vitro, suggesting that wild-type spectral features disrupted in vitro by mutagenesis may be recovered in intact cells. Calibration of intracellular pH (pHi) with in situ fluorescence following excitation in either region revealed a semilogarithmic relationship between fluorescence intensity and pH within the physiologic range. We therefore measured pHi changes attributable to altered Na/HCO3 cotransport (NBC) activity both in GFPmut1-expressing cells and in paired untransfected cells loaded with BCECF. Basal NBC activity was the same in each group, as was the stimulation of activity by 10% CO2, thus validating the utility of GFPmut1 as a fluorescent probe for pHi and establishing a novel, useful, and practical application for GFPmut1 in monitoring pHi in real time.