The effect of hyperthermia on the cytotoxicity of 3-(2-chloroethyl)-2-[(2-chloroethyl)amino]-tetrahydro-2H-,1,3, 2-oxazaphosphorine-2-oxide, ifosfamide (IFO) was investigated in vivo. Tumours were early generation isotransplants of a spontaneous C3Hf/Sed mouse fibrosarcoma, FSa-II. The tumour cell suspensions containing approximately 2 x 10(5) cells were transplanted into the dorsal site of the C3Hf/Sed mouse foot. Hyperthermia was given by immersing the tumour-bearing foot into a constant temperature water bath set at 41.5 degrees C for 0-90 min when tumours reached 34 mm3. IFO was administered i.p. immediately before hyperthermia. Tumour response was studied by the tumour growth (TG) time assay; namely, the TG time or the time for one-half of the treated tumours to reach 700 mm3 from the initial treatment day was determined and the dose-response curves was fitted between the TG time and IFO dose. The anti-tumour effect of IFO was enhanced at this elevated temperature. The thermal enhancement ratio (TER) or the ratio of the slope of dose-response curve at 41.5 degrees C to that of dose-response curve without hyperthermia was relatively small for a short treatment time of 30 min. This TER was smaller for IFO than the TERs for cyclophosphamide (CY) and BCNU which had been studied in our laboratory. However, the TER for IFO increased greatly with a prolongation of treatment time from 30 to 90 min, and exceeded the TER for CY. The TERs were 1.5, 2.6 and 3.6 for heating time of 30, 60, and 90 min, respectively, indicating that a long treatment time such as 90 min at moderately elevated temperatures could result in a substantial enhancement of the antitumour effect of IFO.