We wished to determine if local anesthetics (LAs) induced changes in intracellular free calcium ([fCa2+i]) that could have an effect on cell killing by hyperthermia. Flow cytometry was used to measure [fCa2+i] of mouse NIH 3T3 cells during heating at 45.5 degrees C. In both non-tolerant and thermotolerant cells, heating caused a rapid increase (within 1 min) in [fCa2+i] of approximately 100 nM, which remained relatively constant during 25 min of continued heating; however, survival was higher in thermotolerant cells. Procaine, lidocaine, and tetracaine had no effect on survival or [fCa2+i] of cells kept at 37 degrees C up to 25 min. Cells heated with procaine and lidocaine showed no difference in [fCa2+i] compared to cells heated without LAs but were greatly sensitized to killing. Cells heated with tetracaine became permeable to trypan blue within 10-15 min of heating. We conclude that heat sensitization by LAs does not involve changes in [fCa2+i]. Furthermore, these studies reject the hypothesis that changes in [fCa2+i] are involved in heat-induced cell killing.