Internal pH regulation is vital for many cell functions, including transport mechanisms and metabolic enzyme activity. More specifically, transport mechanisms are to a wide degree governed by internal pH distributions. We introduce the term standard deviation of the intracellular pH (s.d.(pHint)) to describe the internal pH distributions. The cellular pH distributional response to external stress such as heat has not previously been determined. In this study, the intracellular pH (pHi) and the s.d.(pHint) of Saccharomyces cerevisiae cells exposed to supralethal temperatures were measured using fluorescence ratio imaging microscopy (FRIM). An exponential decline in pHi was observed after an initial small decline. For the first time, we report the use of FRIM for determining in vivo plasma membrane proton permeability coefficients in yeast. Furthermore, the exponential decay of pHi and the rupture of the cell plasma membrane, as measured by propidium iodide staining, at 70°C were not simultaneous but were separated by a significant temporal difference. Finally, a nonlinear relationship between the pHi and s.d.(pHint) was found; i.e. the s.d.(pHint) was significantly more sensitive to supralethal temperatures than pHi. s.d.(pHint) is therefore proposed as an early health/vitality indicator in S. cerevisiae cells exposed to heat stress.