The effects of an acidic environment on the induction of apoptosis by 42 degrees C hyperthermia were investigated. An acidic environment (pH 6.6) enhanced the hyperthermia-induced apoptosis in HL-60 human promyelocytic leukemia cells as judged by the DNA fragmentation, flow cytometric analysis of DNA content, and cleavage of poly(ADP-ribose) polymerase. Hyperthermia exerted no effect on the expression of Bcl-2 and Bax, regardless of the environmental acidity during heating. The time of increase in apoptosis after heating coincided with the time of decrease in the G1-phase cell population. It seemed that the increase in heat-induced apoptosis in HL-60 cells in an acidic environment was due to a direct increase in the proteolytic cleavage of poly(ADP-ribose) polymerase by acidic caspases without the involvement of Bcl-2 and Bax, and that heat-induced apoptosis occurred during G1 phase in HL-60 cells.