Confocal spectral imaging (CSI) technique was used for quantitative analysis of the uptake, subcellular localization, and characteristics of localized binding and retention of anticancer agent mitoxantrone (MITOX) within human K562 erythroleukemia cells. The CSI technique enables identification of the state and interactions of the drug within the living cells. Utilizing this unique property of the method, intracellular distributions were examined for monomeric MITOX in polar environment, MITOX bound with hydrophobic cellular structures, naphthoquinoxaline metabolite, and nucleic acid-related complexes of MITOX. The features revealed were compared for the cells treated with 2 microM or 10 microM of MITOX for 1 h and correlated to the known data on antitumor action of the drug. MITOX was found to exhibit high tendency to self-aggregation within intracellular media. The aggregates are concluded to be a determinant of long-term intracellular retention of the drug and a source of persistent intracellular binding of MITOX. Considerable penetration of MITOX in the hydrophobic cytoskeleton structures as well as growing accumulation of MITOX bound to nucleic acids within the nucleus were found to occur in the cells treated with a high concentration of the drug. These effects may be among the factors stimulating and/or accompanying high-dose mitoxantrone-induced programmed cell death or apoptosis.