We have evaluated the combination of the radiosensitizing and bioreductive alkylating agent RSU-1069 with hyperthermia and radiation in an attempt to improve the potential effectiveness of hyperthermia and radiation against locally advanced malignancies. In vitro studies in FSaIIC murine fibrosarcoma cells demonstrated that 1 h exposure to RSU-1069 was more cytotoxic toward hypoxic than normally oxygenated cells at 37 degrees C and pH 7.40 and was only minimally more cytotoxic at hyperthermic temperatures. At pH 6.45, however, RSU-1069 became significantly more toxic toward hypoxic cells and its cytotoxicity was markedly increased at hyperthermic temperatures. In contrast, the ability of this agent to radiosensitize hypoxic FSaIIC cells significantly diminished at pH 6.45. Hoechst 33342 diffusion selected FSaIIC tumor subpopulation studies revealed that hyperthermia and RSU-1069 were more toxic towards dim (hypoxic) cells, while radiation was more toxic towards bright (normally oxygenated) cells. A combination of all three modalities resulted in an equal and significant kill of hypoxic and oxygenated cells. These results suggest that the combination of RSU-1069, local hyperthermia and radiation has considerable clinical potential.