The interaction of the antitumor agent daunomycin (DN) with ferric iron has been analysed by Mossbauer spectroscopy, EPR, extended X-ray absorption fine structure (EXAFS), and magnetic susceptibility measurements. In contrast to literature data, at millimolar iron and anthracycline concentrations no solitary Fe(DN)3 complexes are formed in appreciable amounts. The Mossbauer spectroscopic analysis revealed severe dependencies on temperature, on the preparation procedure, the time allowed for equilibration, and on the metal/ligand ratio. The Mossbauer spectra exhibit two components: a broad magnetic sextet and a quadrupole doublet at an Fe/DN molar ratio of 1 : 3 and exclusively a doublet at a molar ratio of 1 :20, indicating an equilibrium of these two spectral components. The EPR spectra are dominated by a signal at g,,, = 2. Double integration of the EPR signals enabled the determination of their spin density and a correlation between EPR and Mossbauer spectra. The Mossbauer sextet species is EPR invisible and corresponds to magnetically ordered polynuclear aggregates with high magnetic anisotropy. EXAFS and susceptibility measurements provide additional evidence for the formation of polynuclear aggregates of ferric daunomycin. The quadrupole doublet species in the Mossbauer spectra correlates with the g = 2 signal in EPR. This species is also related to a magnetically ordered system, exhibiting, however, superparamagnetic behavior due to less magnetic anisotropy. Since daunomycin forms dimers in aqueous solution at millimolar concentrations, we conclude that the cooperative phenomena observed in EPR and Mossbauer spectra are a consequence of its stacking effects.