The combination of cyclosphosphamide (CP) plus methotrexate (MTX) was found to be therapeutically synergistic against the L1210 ascites tumor. After inoculation with 1 x 10(6) L1210 cells to (C57BL/6 x DBA/2)F1 mice, ip administration of CP (200 mg/kg on Day 5) plus MTX (15 mg/kg on Days 5, 7, 9, and 11) resulted in a significant increase in mean lifespan, compared to optimal treatment with either CP or MTX alone. The contribution of the single, simultaneous dose of CP plus MTX on Day 5 to therapeutic synergism was examined. The combination of CP plus MTX on Day 5 produced a greater decrease in tumor cell numbers than either drug alone. Measurements of the time course of inhibition and recovery of 3H-deoxyuridine incorporation into DNA showed that within 48 hours, recovery of DNA synthesis in small intestine and bone marrow was almost complete after either CP, MTX, or CP plus MTX. In contrast, tumor, which had recovered within 48 hours after MTX, still remained greater than 90% inhibited after eight CP or CP plus MTX. Measurements of bone marrow nucleated cellularity after therapy showed that the single, simultaneous dose of CP plus MTX was no more toxic to bone marrow than CP alone, although it was more toxic than MTX alone. No alterations in the distribution of either MTX or alkylating metabolites of CP could be detected in tumor or normal tissues when CP and MTX were administered simultaneously.