1,6-Diphenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene are fluorophores used to explore different hydrophobic domains of membrane bilayers (Andrich, M.P. and Vanderkooi, J.M. (1976) Biochemistry 15, 1257-1265; Prendergast, F.G., Haugland, R.P. and Callahan, P.J. (1981) Biochemistry 20, 7333-7338). Fluorescence resonance energy transfer between these fluorophores, acting as energy donors, and the anthracycline, daunomycin, as the acceptor, was used to analyze the interaction of the drug with natural membranes, and its relative location within the membrane bilayer. The transfer process was demonstrated by: (1) emission fluorescence of the acceptor when the samples were excited at the excitation maximum of the donor (360 nm); and (2) progressive quenching of the energy donor (at 428 nm) when in the presence of increasing acceptor concentration. Also, the disruption of the energy transfer by solubilization of the membrane with Triton X-100 evidences a role for the membrane in providing the appropriate site(s) for energy transfer to occur. At moderately low daunomycin/membrane lipid ratios, the different efficiencies of resonance energy transfer between the two donors and daunomycin predicts a preferential, but not exclusive, location of the drug at membrane 'surface' domains, i.e., those regions of the bilayer explored by the 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probe. In support of this observation, a large fraction (approx. 75%) of membrane-associated daunomycin was rapidly sequestered away from the membrane upon addition of excess DNA, which forms high-affinity complexes with daunomycin (Chaires, J.B., Dattagupta, n. and Crothers, D.M. (1982) Biochemistry 21, 3927-3932), thus acting as a drug 'sink'. Also, a large fraction of drug was accessible to fluorescence quenching by iodide, a collisional water-soluble quencher. On the other hand, a smaller population of the membrane-associated daunomycin was characterized by slow sequestering by the added DNA and inaccessibility to quenching by iodide. We conclude that the daunomycin, which is only slowly sequestered, is located deep within the hydrophobic domains of the bilayer, likely to be those probed by 1,6-diphenyl-1,3,5-hexatriene.