Although changes in both pH<inf>in</inf> and [Ca<sup>2+</sup>]<inf>i</inf> have been observed in response to a variety of agonists, it is not clear whether these ionic events work independently or are coordinated to lead to a specific physiological response. One of the fundamental problems in studying these ionic events is that changes in pH<inf>in</inf> modify Ca<sup>2+</sup> regulatory mechanisms and changes in Ca<sup>2+</sup> may modify pH regulation. It is desirable to use a technique that allows concomitant monitoring of these two ions in cell populations with high time resolution. Furthermore, like many Ca<sup>2+</sup> binding proteins, all Ca<sup>2+</sup> -sensitive fluoroprobes are inherently sensitive to pH owing to competition of H<sup>+</sup> for the Ca<sup>2+</sup> -binding sites. This chapter describes experimental paradigms that provide optimum conditions for simultaneous measurement of pH from the fluorescence emission of snarf-1, and Ca<sup>2+</sup> using fura-2. The fluorescence spectra of these compounds are sufficiently different to allow simultaneous measurement of pH and Ca<sup>2+</sup> both in vitro and in vivo. Moreover, the ratio of the H + - sensitive wavelengths of snarf-1 is unaffected by Ca<sup>2+</sup> , or the concomitant presence of fura-2 in cells. Although the fluorescence ratio of fura-2 is insensitive to the presence of snarf-1, it is affected by pH, as indicated above. We describe procedures to correct for this effect and to obtain calibration parameters for fura-2 and snarf-1 required to facilitate analysis of pH and Ca<sup>2+</sup> concentrations within cell populations.