The effect of a short-term energy deprivation (ischemia) on thermoresistance and heat-shock protein (HSP) synthesis in murine ascites EL-4 thymoma cells was studied in vitro. The incubation of the cells in glucose-free medium with rotenone (respiratory inhibitor) for 10 min caused rapid ATP depletion (to 9% of the initial level). After recovery, the synthesis of HSP70 and HSP90 was stimulated in the cells and they became greatly more resistant to hyperthermia than the control cells. The simultaneous rotenone and thermal treatment significantly decreased cell viability. The transition of HSP70 to Triton X-100-insoluble cell fraction was found in the ATP-depleted cells as well as in the heat-shocked cells, and 1 mM ATP fully reversed such insolubilization when it was added in Triton extraction buffer. The data obtained reveal that transient ATP depletion per se is sufficient to result in the HSP70 insolubilization, thus being conducive to induction of HSP synthesis and thermotolerance in the cells which recovered after energy deprivation. A novel mechanism of protein aggregation in ATP-deficient cells and a possible role of transient ischemia in development of tumor thermotolerance in vivo are discussed.