A ratio-fluorescence assay was developed for on-line localization and quantification of lipid oxidation in living cells. The assay explores the oxidative sensitivity of C11-BODIPY(581/591). Upon oxidation, the fluorescence of this fluorophore shifts from red to green. The probe incorporates readily into cellular membranes and is about twice as sensitive to oxidation as arachidonic acid. Using confocal microscopy, the cumene hydroperoxide-induced oxidation of C11-BODIPY(581/591) was visualized at the sub-cellular level in rat-1 fibroblasts. Preloading of the cells with tocopherol retarded this oxidation. The data demonstrate that C11-BODIPY(581/591) is a valuable tool to quantify lipid oxidation and anti-oxidant efficacy in single cells.