The interaction of doxorubicin with nuclei isolated from rat liver and kidney was studied by fluorospectrometry . The nuclei had at least two different types of binding sites for the drug. Both Mg2+ and Ca2+ competitively inhibited the binding of doxorubicin to the nuclei, which showed a remarkable temperature dependency. No significant difference was observed between the numbers of binding sites (n = 6.70 X 10(-2) mol/mol of DNA for liver; 6.41 X 10(-2) mol/mol of DNA for kidney) or the affinity constants (Ka = 4.85 X 10(5) M-1 for liver; 5.41 X 10(5) M-1 for kidney) under quasi-physiological conditions. These results obtained from in vitro binding experiments support previous suggestions that the differences in the in vivo distribution of doxorubicin among tissues are not due to differences in the nuclear binding of the drug. The amount of nuclei per gram of tissue is the primary determinant of the characteristic tissue distribution of doxorubicin.