The chemo- and radioprotectant drug amifostine (Ethyol; MedImmune, Inc, Gaithersburg, MD) is approved for intravenous (IV) administration; however, the subcutaneous (SC) route is being explored as a practical alternative. We have previously reported equivalence between IV and SC administration using a rat model of radioprotection and active metabolite (WR-1065) tissue pharmacokinetics. To examine the more clinically relevant fractionated and hyperfractionated radiation schedules and the effects of variations in the time of amifostine administration, we expanded these studies to include radioprotection and pharmacokinetic studies of WR-1065 using multiple dosing. To measure radioprotection using a fractionated radioprotection model, rats were given amifostine over a 1-week period at various doses (25 mg/kg, 50 mg/kg, 100 mg/kg; or 162.5 mg/m(2), 325 mg/m(2), 650 mg/m(2), respectively) IV or SC daily 30 minutes before exposure to 7.5 Gy/dose. Rats were fully protected from mucositis at the highest amifostine dose, with protection diminishing as the amifostine was decreased. Equivalent protection was observed whether the drug was given IV or SC. When the number of days of amifostine administration was reduced, protection was diminished. Amifostine also protected against radiation delivered using a 1-week hyperfractionated schedule (4.5 Gy/exposure twice daily), with optimal protection occurring when the drug was administered bid 30 minutes before each exposure (50 mg/kg) or every day before the morning exposure (100 mg/kg). The need for daily dosing to achieve optimal radioprotection was consistent with the tissue pharmacokinetics of the active metabolite. We found that WR-1065 did not accumulate in tissues or in SC-implanted tumors when amifostine was administered daily for 3 weeks. In addition, tissue and tumor levels of WR-1065 declined to baseline 24 hours after each amifostine dose. In a monkey pharmacokinetic model, plasma levels of WR-1065 (characterized by a pronounced spike of WR-1065 immediately after IV administration that was absent when the drug was given SC) were similar to those of humans; however, levels of WR-1065 in the tissues were higher 30 minutes following SC administration and were equivalent 60 minutes following IV or SC administration. These results suggest that maximum tissue levels and protection occur when amifostine is given 30 to 60 minutes before radiation exposure, that treatment breaks reduce the radioprotection by amifostine, and that protection from hyperfractionated radiation is dependent on amifostine dose and schedule.