We have evaluated the pyrene-based ratiometric fluorescent dye, 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS), by using it in conjunction with glass pH-sensitive microelectrodes to measure intracellular pH (pHi) in voltage-clamped snail neurones. Intracellular acidification with propionic acid, and alkalinization following the activation of H+ channels allowed the calibration of the dye to be compared with that of the pH microelectrode over the pH range 6.50-7.50. HPTS calibrated in vitro and glass pH-sensitive microelectrodes produced similar absolute resting pHi values, 7. 16+/-0.05 (n=10) and 7.17+/-0.06 (n=9) respectively in nominally CO2/HCO3--free saline. At both extremes of the pH range there were small discrepancies. At acidic pHi, 6.87+/-0.09 (n=5), the intracellular HPTS measurement differed by -0.08+/-0.03 pH units from the pH-sensitive microelectrode measurement. At alkaline pHi, 7. 32+/-0.10 (n=5), HPTS measurements produced pH values that differed by +0.07+/-0.04 pH units from those of the pH-sensitive microelectrode. Some of the discrepancy could be accounted for by the slow response of the recessed-tip pH-sensitive microelectrode (time constant 77+/-15 s, n=3). Further experiments showed that HPTS, used at an intracellular concentration of 200 microM to 2 mM, did not block activity-dependent pHi changes. The intracellular HPTS concentration was calculated by measurement of intracellular chloride during a series of HPTS-KCl injections. Comparison of HPTS with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), at the same concentration, showed that HPTS produces a larger change in ratio over the pH range 6.00-8.00.