Hyperthermia (temperatures greater than or equal to 42 degrees C) is used clinically to improve the effectiveness of radiation therapy and, although therapeutic gains have been reported, efficacy is limited when tumours are large and/or radiation tolerance is reduced. In order to improve the utility of the hyperthermia/radiation combination we have tested the addition of cisplatin (CDDP) in the laboratory and in the clinic. Our clinical studies have shown that the CDDP/hyperthermia/radiation combination is tolerable and effective, but laboratory investigations demonstrated a relative lack of cytotoxicity in the hypoxic tumour subpopulation. In order to improve the effectiveness of the CDDP/hyperthermia/radiation combination against hypoxic cells we have evaluated the addition of mitomycin C, a hypoxic cell cytotoxic agent to this combination. Mitomycin C (5 mg/kg) i.p. produced a tumour growth delay (TGD) of about 5.3 days in the FSaIIC murine fibrosarcoma; hyperthermia (43 degrees C x 30 min) caused only about 1.4 day TGD and the combination of mitomycin C followed immediately by hyperthermia caused a TGD of about 8.6 days. CDDP (5 mg/kg) i.p. followed by hyperthermia and then 3 Gy on day 1 only of a 5 day x 3 Gy radiation protocol produced a TGD of about 25 days. With the addition of mitomycin C just before CDDP a TGD of about 44 days resulted. Whole tumour excision experiments demonstrated that mitomycin C was highly interactive with CDDP at 37 degrees C and was dose-modifying. When used with CDDP and hyperthermia, however, mitomycin C added little additional cytotoxicity. Hoechst 33342 dye diffusion-determined tumour subpopulation studies indicated a marked effect of the addition of mitomycin C in the dim (enriched in hypoxic cells) subpopulation and nearby equal cytotoxicity in both bright (enriched in euoxic cells) and dim cells resulted. These investigations suggest considerable potential therapeutic efficacy to the addition of mitomycin C to the CDDP/hyperthermia/radiation combination.