Lipofuscin pigment determination in tissue extracts was quantitated by the use of its property of fluorescence. Chloroform: methanol tissue extracts were purified on Sephadex LH-20 columns before quantitative fluorescence measurements of the lipofuscin pigments. Interfering compounds separated by chromatography were retinol and a lower mol wt fluorescent compound. Irradiation of tissue extracts with ultraviolet light was not sufficient to eliminate the interference caused by retinol and the lower mol wt compound. Purified lipofuscin pigments from blood, lung, liver, spleen, brain, heart, and kidney tissues demonstrated distinct fluorescent emission maximum at 435 nm and excitation maxima between 345 and 350 nm.