Enhanced cell kill has been observed when experimental tumors were treated with alkylating agents in combination with 2-nitroimidazoles (2-NI). In this study, modification of the cell kill induced by cyclophosphamide (CY) and an analog, ifosfamide (IFO), by two radiation sensitizers, misonidazole (MISO) and etanidazole (SR-2508), was measured. Three important parameters were determined: (a) the necessity for hypoxic reduction of the 2-NI to achieve an increase in tumor cell kill, (b) the optimal timing for administration of the alkylating agents and the 2-NI, and (c) the degree of enhancement of the CY- and IFO-induced cell kill. The subcutaneous (sc) 9L tumor model in male Fisher 344 rats was used in these experiments, and the endpoint measured was clonogenic cell survival 18-20 hr after treatment. Under hypoxic conditions, MISO potentiated both CY- and IFO-induced cell kill with a sensitizer enhancement ratio of approximately 1.3 and 1.5, respectively, at the 10(-3) survival level. This enhancement was seen when CY was administered simultaneously or 1.5 hr prior to MISO administration. A similar enhancement of CY-induced cell kill was measured under hypoxic conditions when SR-2508 was used. Enhanced IFO-induced cell kill was measured under hypoxic conditions only when the IFO was given 1 hr before MISO administration. No enhancement of the IFO-induced cell kill was observed when SR-2508 was used instead of MISO. Increased normal tissue damage (i.e., hemorrhagic cystitis) was observed when the MISO was administered along with CY or IFO. Four conclusions can be drawn from these data. Metabolism of the 2-NI by hypoxic cells is necessary for potentiation of CY- or IFO-induced cell kill. Only MISO can potentiate the cell kill induced by IFO. The timing of administration of the alkylating agents and the 2-NI is a critical determinant of the extent of the cell kill obtained. Cell kill induced by IFO appears to be enhanced by MISO to a greater extent than the cell kill induced by CY.