A quantitative fluorescent microscopy system was developed to characterize, in real time, the effects of supraphysiological temperatures between 37 degrees and 70 degrees C on the plasma membrane of mouse 3T3 fibroblasts and isolated rat skeletal muscle cells. Membrane permeability was assessed by monitoring the leakage as a function of time of the fluorescent membrane integrity probe calcein. The kinetics of dye leakage increased with increasing temperature in both the 3T3 fibroblasts and the skeletal muscle cells. Analytical solutions derived from a two-compartment transport model showed that, for both cell types, a time-dependent permeability assumption provided a statistically better fit of the model predictions to the data than a constant permeability assumption. This finding suggests that the plasma membrane integrity is continuously being compromised while cells are subjected to supraphysiological temperatures.