Purpose: We tested therapeutic efficacy of two dose painting strategies of applying higher radiation dose to tumor subvolumes with high FDG uptake (biologic target volume, BTV): dose escalation and dose redistribution. We also investigated whether tumor response was determined by the highest dose in BTV or the lowest dose in gross tumor volume (GTV). Experimental Design: FDG uptake was evaluated in rat rhabdomyosarcomas prior to irradiation. BTV was defined as 30% of GTV with the highest (BTVhot) or lowest (BTVcold) uptake. To test efficacy of dose escalation, tumor response (time to reach two times starting tumor volume, TGT(V2)) to Hot Boost irradiation (40% higher dose to BTVhot) was compared with Cold Boost (40% higher dose to BTVcold), while mean dose to GTV remained 12 Gy. To test efficacy of dose redistribution, TGT(V2) after Hot Boost was compared with uniform irradiation with the same mean dose (8 or 12 Gy). Results: TGT(V2) after 12 Gy delivered heterogeneously (Hot and Cold Boost) or uniformly were not significantly different: 20.2, 19.5, and 20.6 days, respectively. Dose redistribution (Hot Boost) with 8 Gy resulted in faster tumor regrowth as compared with uniform irradiation (13.3 vs. 17.1 days; P = 0.026). Further increase in dose gradient to 60% led to a more pronounced decrease in TGT(V2) (10.9 days; P <0.0001). Conclusions: Dose escalation effect was independent of FDG uptake in target tumor volume, while dose redistribution was detrimental in this tumor model for dose levels applied here. Our data are consistent with the hypothesis that tumor response depends on the minimum intratumoral dose.