Purpose: To quantify the reduction of radiation-induced oral mucositis by amifostine as a function of administration route. Material and Methods: Mucosal ulceration of lower mouse tongue epithelium was analyzed. Amifostine was injected at 1.8 mg/ injection subcutaneously (s.c.) or intravenously (i.v.), 45 min or 10 min prior to irradiation. With single-dose irradiation, a single amifostine injection was given. During daily fractionated irradiation (5 x 3 Gy) for 1 week, amifostine was administered s.c. or i.v. twice (days 0, 3), or s.c. on all irradiation days (days 0-4). With ten fractions over 2 weeks, five s.c. injections were given in week 1 (days 0-4) or week 2 (days 7-11), or both. Two i.v. injections were given either in week 1 (days 0, 3) or week 2 (days 7, 10). All fractionation protocols were terminated by graded test doses to generate full dose-effect curves. Results: In a single-dose control experiment, the ED₅₀ (dose after which ulcer induction is expected in 50% of the mice) was 11.7 ± 1.4 Gy. Intravenous application of amifostine increased the ED₅₀ to 14.0 ± 1.4 Gy (p = 0.024), while s.c. administration had no significant effect. The ED₅₀ for test irradiation after 5 x 3 Gy was 5.8 ± 1.4 Gy. Two s.c. or i.v. amifostine injections yielded ED₅₀ values of 7.2 ± 1.1 Gy (p = 0.0984) or 7.6 ± 1.2 Gy (p = 0.0334); five s.c. injections increased the ED ₅₀ to 8.2 ± 0.9 Gy (p = 0.0039). The ED₅₀ after 10 x 3 Gy/2 weeks was 6.6 ± 1.8 Gy. Subcutaneous or intravenous administration of amifostine in week 1 yielded a significant increase in ED ₅₀ to 9.4 ± 2.5 Gy (p = 0.0099) and 10.0 ± 2.2 Gy (p = 0.0014). By contrast, amifostine administration in week 2 had no significant effect. Administration in weeks 1 and 2 resulted in an ED₅₀ of 10.8 ± 3.6 Gy (p = 0.0053). Conclusion: Amifostine during daily fractionated irradiation is effective only if administered in the initial treatment phase, i.e., week 1 in the mouse. The differences in the effect in weeks 1 and 2 suggest mechanisms of action other than radical scavenging.