Background and purpose: Aim of this study was to assess the impact of the administration protocol of palifermin on amelioration of oral mucositis after fractionated irradiation. Materials and methods: Mouse tongue ulceration was analysed as the clinically relevant endpoint. Daily fractionated irradiation (5×3 Gy/week, days 0 to +4, +7 to +11, with a weekend gap on days +5 and +6) was followed by graded test doses on day +14, i.e. after a second weekend gap. Palifermin (5 mg/kg) was injected subcutaneously. In the first series of experiments, the effect of three daily injections (days -3, -2 and -1) was compared with a single administration either on day -2 or -1; all animals received a further injection on day +4. In the second series, a single or three injections were given in the weekend gap between fractionated irradiation (days +5 to +6), with an additional administration on day +11. In a final protocol, single weekly injections of palifermin were given either on days -3, +4 and +11, days +4, +11 and +18, or on days -3, +4, +11 and +18. Results: The ED50 (dose after which ulcer induction is expected in 50% of the mice) to single dose irradiation was 11.5±0.7 Gy. The ED50 for test irradiation after 10×3 Gy was 5.7±1.6 Gy. Palifermin administration before the start of fractionated irradiation and on day +4 increased the ED50 to 10-12 Gy, administration over the first weekend and on day +11 to 11-15 Gy. Administration over three consecutive weekends, starting on day -3 or day +4, increased the ED50 to 13.0±0.1 and 14.9±0.3 Gy. Single weekly KGF administrations over four weekends, including the weekend prior to and after completion of radiotherapy, showed no further increase in ED50. Conclusions: A single palifermin injection during the weekend gap before or during fractionated irradiation is as effective as three applications. Onset of the palifermin treatment during the first weekend gap between fractionated irradiation is more effective than during the weekend before radiotherapy. The effect of palifermin on oral mucositis can be increased by three weekly injections, while four injections do not yield a further increase in ED50.