Hyperthermia causes a large (three-to fivefold) increase in intracellular free calcium ([Ca2+]i) in HA-1 fibroblasts. Increased [Ca2+]i appears initially to be due to release of Ca2+ from an internal store, probably located in the endoplasmic reticulum. A subsequent influx of Ca2+ from the extracellular medium is then observed. These heat-induced changes in Ca2+ homeostasis are correlated with turnover of the phosphoinositides (PI), a class of phospholipids whose metabolism has been shown to regulate Ca2+ in a wide variety of cells (M. J. Berridge and R. F. Irvine, Nature 312, 315 (1984]. Hyperthermia induces rapid release of inositol 1,4,5-trisphosphate (IP3) within 1 min at 45 degrees C; IP3 release precedes the heat-induced rise in [Ca2+]i. IP3 release, a result of phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C, is the initial step in PI turnover. Later accumulation of phosphatidic acid, another metabolite in the PI pathway, is correlated with the delayed, heat-induced influx of 45Ca2+ from the extracellular environment. The data thus indicate that heat-induced changes in Ca2+ homeostasis are correlated with activation of PI turnover. They indicate that this class of lipids may be closely involved in heat-induced changes in cellular Ca2+ homeostasis. Cell Ca2+ appears to be important in some aspects of the cellular response to heat.