Lipophilic analogues of dipyrrometheneboron (BODIPY-FL) dyes, used for membrane studies, normally fluoresce in the green wavelength region (approximately 516 nm), but at high local concentration, they shift their emission to the red region (approximately 540-600 nm) via excimer formation. A two-wavelength-based method is described that utilizes the excimer-forming property of BODIPY-FL for the sensitive monitoring of membrane lipid peroxidation on erythrocytes (RBCs). Bodipy-FL- C3-EDA, a relatively water-soluble analogue of BODIPY-FL, loses its single fluorescence peak at 516 nm upon reaction with peroxyl radicals generated in aqueous phase by 2,2'-azobis(2-amidinopropane), AAPH, and the loss of fluorescence is prevented by the presence of the peroxyl radical scavenger, Trolox (a water-soluble analogue of vitamin E). Hexadecanoyl-BODIPY-FL (C16-BODIPY, a lipophilic analogue of BODIPY-FL) incorporated into RBC membranes, in addition to the peak at 516 nm, also forms excimer fluorescent peaks at 546 and 590 nm. The relative intensity of the emission peaks depends on the concentration of membrane-incorporated dye. Upon addition of cumene hydroperoxide (CH, 0-10 microM) or benzoyl peroxide (BP, 0-5 microM) to C16-BODIPY-labeled RBC suspensions, gradual changes in the fluorescent peaks occur, the 516 nm peak initially increases, then decreases, while the 546 and 590 nm excimer peaks continuously decrease, as measured by fluorometry or by flow cytometry. The data indicate that lipid peroxidation radicals reacting with C16-BODIPY localized on RBC membranes oxidize the dye and the resulting molecule cannot participate in the excimer formation; this oxidization leads to the observed changes in the fluorescent peaks. The ratio of the fluorescence levels at 590 and 516 nm is a measure of the excimer formation between fluorophores and can be used to monitor the onset of lipid peroxidation in RBCs.