Studies were performed to synchronize L-1210 tumor cells in S [DNA synthesis] phase in an effort to maximize the effect of subsequent pulse dose chemotherapy. Hydroxyurea (HU) was administered by continuous i.v. infusion 5 days after tumor implantation. Perturbation effects on the S-phase cells were measured by serial tritiated thymidine labeling indices. Effects on cell-cycle progression were measured by DNA content distribution analysis using flow cytometry. Synchronization of tumor cells was achieved with HU infusion (48 mg/kg/hour .times. 24 h), resulting in 90% of the cells in S-phase. Following infusion, synchronous progression of S-phase cells into G2/M [post synthetic period/mitosis] and then G1 presynthetic period was apparent from +0 to +10 h later. The susceptibility of HU-synchronized cells to subsequent chemotherapy was determined by treating mice with cytosine arabinoside (Ara-C), methotrexate (MTX) or adriamycin (ADR) pulse doses at various intervals following infusion. Synergy, measured by prolongation of survival times, resulted when Ara-C was administered immediately after the end of the infusion. Survival times then decreased as the fraction of cells in S phase decreased. The survival times of mice treated with MTX or ADR pulse doses after HU infusion were additive at best and did not correlate with fluctuations in the S phase compartment. Prior synchronization of tumor cells in S phase was therapeutically advantageous when coupled with appropriately timed Ara-C pulse doses. There was little advantage in combining HU infusion with subsequent MTX or ADR therapy.