The effects of supranormal temperature on the activity of poly(ADP-ribose) glycohydrolase were studied by assaying the enzyme in cell extracts derived from cells subjected to hyperthermia and comparing with extracts that were heated in vitro. The enzyme activity was reduced by both hyperthermic treatment of cells and by heating of cell extracts; however greater reductions were observed when intact cells were subjected to hyperthermia. The additional reduction observed when intact cells were heated was reversed when cells were allowed to recover at 37 degrees C following hyperthermia. We postulate that hyperthermia alters poly(ADP-ribose) glycohydrolase activity by two mechanisms, an irreversible thermal denaturation of the enzyme and a reversible metabolic alteration. Changes in poly(ADP-ribose) glycohydrolase activity can account in full for the observed alterations of poly(ADP-ribose) metabolism that occur following hyperthermia.