Although the mammalian heat shock response has been well characterized, the processes that mediate the induction of the response and the regulation of heat shock protein function are not completely understood. We have investigated the potential role in heat-shocked cells of phosphoinositide-specific phospholipase C (PLC), a membrane enzyme activity involved in transmembrane signal transduction. Our studies indicate that heat shock activates PLC in each of seven cell lines, including cells of human, rat, mouse, and hamster origin. Heat shock produced increases in inositol phosphate concentrations comparable in magnitude to those achieved after simulation with growth factors, indicating that heat shock might initiate transmembrane signaling cascades of potential importance in cellular regulation. Common cellular responses to heat and growth factors also included feedback modulation of PLC by its products and the parallel stimulation of phospholipase A2 activity. In addition to heat shock, other agents that induce the stress response stimulated PLC activity. The data indicate a close correlation between expression of the mammalian heat shock response and stimulation of PLC activity and indicate a possible role for this enzyme activity in the regulation of some aspects of the stress response.