The influence of hyperthermia and environmental pH on proteolytic activity was studied in murine ascites tumor cells in vitro. PNJ ascites tumor cells were incubated with [125I]cytochrome c at 42.5 or 37 degrees C in a modified Krebs-Ringer buffer adjusted to pH 7.2 or 6.4. Incubation at normal temperature at pH 6.4 and 7.2 or at 42.5 degrees C and pH 7.2 resulted in identical protein digestion. However, hyperthermic incubation at pH 6.4 resulted in a significant increased activity. This was also observed after only 1 hour of hyperthermic incubation followed by subsequent incubation in an acidic environment at normal temperature. The increased proteolytic activity following hyperthermic treatment under acidic conditions may support the hypothesis that increased lysosomal activity is of primary importance in the hyperthermic tumor-cell destruction in vivo.