Combining radiofrequency (RF) ablation with i.v. liposomal doxorubicin (Doxil) increases intratumoral doxorubicin accumulation and tumor destruction. The purpose of this study was to characterize and better define the specific parameters of such treatment in an animal tumor model. Four hundred R3230 mammary adenocarcinoma nodules were implanted in 250 Fischer rats. First, paired tumors received combined standardized RF (70°C ± 2°C, 5 min) followed 30 min later with i.v. Doxil (1 mg) or Doxil alone. Intratumoral doxorubicin uptake was evaluated using fluorospectrophotometry 2–120 h after therapy (n = 110). The effects of varying i.v. Doxil doses (0.0625–7.0 mg; n = 100) and the RF tip temperatures (45°C–90°C; n = 190) on subsequent intratumoral doxorubicin uptake and induced tumor necrosis were evaluated. Intratumoral doxorubicin accumulation increased to a maximum at 72 h with greater uptake in the RF-ablated tumors compared with controls (P < 0.01). Greater dose-dependent intratumoral doxorubicin increases (to 37.3 ± 7.7 μg/g) were seen with combined RF/Doxil therapy (P < 0.01). RF ablation reduced the i.v. Doxil dose needed to achieve intratumoral doxorubicin uptake of 13 μg/g from 7 to 2 mg. Increasing tip temperatures from 50°C to 90°C increased the ratio of doxorubicin in RF to nonablated tumors from 1.2 ± 0.4 to 5.9 ± 3.8 (P < 0.01). At all temperatures, greater tumor necrosis was identified for RF/Doxil-treated tumors compared with tumors treated with RF alone (P < 0.05). The threshold for inducing necrosis was 5°C lower for tumors receiving combined therapy (P < 0.01). RF tumor ablation sharply increases intratumoral Doxil accumulation over i.v. Doxil alone, enabling a reduction of systemic dose while obtaining higher intratumoral concentrations than otherwise achievable. Combined therapy also increases tumor destruction over either therapy alone.