We describe a new procedure for the preparation of liposomal doxorubicin. Doxorubicin can be efficiently complexed to preformed or lyophilized cardiolipin-containing liposomes. Complex formation was performed by vigorous vortexing. As much as 96.8% of the initial drug quantity may be bound to those liposomes under optimal incubation conditions (4 h at 37 degrees C). The binding study showed the presence of two levels of specific binding (dissociation constants, 28 +/- 8 microM and 1.0 +/- 0.3 mM). The drug is firmly integrated in the liposome-membrane lipid bilayer rather than binding at the surface. Cytotoxicity studies using tumor cells revealed efficient drug delivery using liposome-complexed doxorubicin. This new liposomal doxorubicin preparation reverses multidrug resistance in MCF-7/ADR and CH LZ cells at levels equivalent to that obtained with a previously described liposome-encapsulated doxorubicin preparation, showing that the drug is integrated as well in the liposome carrier and is transported as well into cells. Increased concentration of liposomes at the subcytotoxic level in liposome-complexed doxorubicin enhances drug cytotoxicity in multidrug-resistant CH LZ cells as compared with liposome-encapsulated drug. This new preparation for liposomal doxorubicin may be carried out immediately prior to clinical administration, offering advantages in terms of cost and stability.