Improved immunocytochemical (ICC) detection of the anthracycline anticancer antibiotic daunomycin (DM) has been achieved by used of hydrogen peroxide oxidation prior to ICC staining for DM. The new method greatly enhanced the localization of DM accumulation in cardiac, smooth and skeletal muscle of rats after a single i.v. dose of the drug. DM accumulated in the nuclai as well as in the sarcoplasm, where it occurred in the form of small granules, which were particularly evident in cardiac muscle cells. The distribution of the granules coincided with that of mitochondria. Uptake of DM in nuclei and mitochondria of heart muscle cells may help to improve our understanding of the cardiac toxicity of DM and related anthracyclin antibiotics. A number of ELISA tests were carried out in order to elucidate the mechanisms of H₂O₂-assisted antigen retrieval. A possible mechanism is that DM is reduced and converted to its semiquinone and/or hydroquinone derivative in vivo. Oxidation by hydrogen peroxide acts to convert these derivatives back to the native antigen. The improved ICC methodology using oxidation to recreated native antigens from reduced metabolites may be helpful also with respect to the localization of other drugs.