Two biochemically different rescue agents, citrovorum factor (CF) and thymidine-inosine-allopurinol (TIA), were compared in an attempt to identify the mechanism for the increased therapeutic index achieved with high-dose methotrexate (MTX) plus rescue. Both CF and TIA were capable of protecting mice from MTX dosages up to 2000 mg/kg. Treatment of L1210-bearing mice with 2000 mg/kg MTX plus CF or TIA produced a 70 and 100% increase in life span, respectively, compared with 29% increase in life span achieved with the maximally tolerated dose of MTX alone. Bioassay of surviving peritoneal L1210 cells showed that a 4.5-log tumor kill occurred 24 hr after 2000 mg/kg MTX, while 400 mg/kg MTX produced only a 2-log cell kill. This differential tumor kill in the 4-hr period after MTX and prior to the onset of rescue accounted for the observed increase in animal survival times. In addition, treatment with 2000 mg/kg MTX resulted in a one-log-greater tumor kill of cells metastasized to the brain than did treatment with 400 mg/kg MTX. Following 2000 mg/kg MTX, additional tumor kill, as measured by bioassay, occurred during the period of TIA rescue but not during CF rescue, which was consistent with the observed differences in survival times between CF- and TIA-rescued mice. DNA synthesis in tumor and host tissue, as measured by the rate of [3H]dCyd incorporation into DNA, was cyclic after TIA administration but not after CF administration. The cyclic nature of DNA recovery in TIA-treated mice paralleled plasma kinetics of thymidine. It is postulated that " thymineless " intervals created by the rapid disappearance of thymidine resulted in inhibition of DNA synthesis and additional tumor cell kill during TIA rescue. Normal tissue did not appear to be adversely affected by exposure to these " thymineless " intervals.