The mechanism of interorgan and interspecies variations in the tissue distribution of doxorubicin was studied in rats, rabbits, and guinea pigs. The permeation properties of doxorubicin were investigated by using isolated rat erythrocytes as a model membrane. A significant dependency of the uptake rate constant on medium pH was observed, suggesting that only un-ionized doxorubicin is diffusible through the plasma membrane. The values of plasma unbound fraction (fp) of doxorubicin were 0.344 for rats, 0.415 for rabbits, and 0.529 for guinea pigs. The tissue DNA concentrations of rats were larger than those of rabbits and smaller than those of guinea pigs in corresponding organs or tissues. An in vitro organ model that described the distribution behavior of doxorubicin in the whole body was constructed, and an equation was derived to estimate the tissue-to-plasma partition coefficients, (Kp) from in vitro experiments. The in vitro Kp values showed comparatively good agreement with the in vivo Kp values for the various organs or tissues in all three species. Doxorubicin is exclusively bound to the nuclei in the cells. The variation of the Kp values in different organs depended on the amount of nuclei per gram of tissue. The primary determinant of the interspecies variation in the Kp values was the difference in tissue DNA concentrations among rats, rabbits, and guinea pigs, and a secondary determinant was the difference in fp values.