Several hundred thousand men receive chemotherapy each year; many are sterilized by this treatment. Temporary testicular circulatory isolation (TCI), a regional drug delivery approach to circumvent this, decreases doxorubicin-induced testicular injury in the rat and provides partial protection from doxorubicin-related infertility. We evaluated the distribution of doxorubicin and its metabolites (doxorubicinol and doxorubicin aglycone) in rats treated with TCI. In each of 56 male Sprague-Dawley rats, the left spermatic cord and gubernaculum were mechanically clamped for 45 minutes. Immediately after clamp application, these rats received doxorubicin (6 mg/kg, intravenous bolus) and were killed at seven time points after doxorubicin administration, ranging from 30 minutes to 48 hours. Twenty-one control rats were treated identically but did not receive TCI. Doxorubicin and its metabolites were extracted from tissue (left testis, right testis, left kidney, heart, left lung, liver) and serum and analyzed by high-performance liquid chromatography. In the TCI group, the distribution of the parent drug and doxorubicinol in tissue and serum closely approximated levels from doxorubicin-treated controls not receiving TCI in all organs except left testis. No anthracycline was detected at any time point in the left testis of the TCI group. These results indicate that TCI completely protects the testis from doxorubicin exposure in this model and that TCI does not affect distribution of doxorubicin in other organs.