There is a close linkage between intracellular pH changes and transmembrane calcium fluxes in many cell types. To understand these associations better it is often desirable to measure pH and [Ca²⁺] simultaneously at a sub-cellular level. Here we report that a mixture of HPTS and Fura-red loaded into a neurone can allow such measurements to be made on a confocal microscope. Intracellular pH was calculated from the ratio of HPTS fluorescence (500-580 nm) when excited with alternating 405 nm and 488 nm light. Fura-red has a much larger Stoke’s shift and fluoresces at >600 nm, and when excited with alternating 405 nm and 488nm light produces a ratio indicating [Ca²⁺]. We find that a mixture of 125 µM HPTS and 62.5 µM Fura-red, when loaded into whole-cell patch-clamped neurones, allows both calcium and pH to be monitored simultaneously in sub-cellular regions with no discernable cross-talk between the two dyes (see Figure 1). The usefulness of this technique may depend upon the type of confocal, and the available filters. Our Leica SP5, with its electronically tuneable beam splitter and spectral emission, allows the two dyes to be separated. However, experiments on our Zeiss LSM510, using just 488 nm excitation, show some overlap between the two dyes. The calcium- and pH-sensitive images are acquired synchronously, using identical wavelengths for the excitation; this allows us to rule out some artefacts when comparing calcium and pH.