A simple, sensitive and rapid spectrofluorimetric method to determine velnacrine, a cholinesterase inhibitor, has been developed and validated. The influence of pH, temperature, ionic strength, presence of excipients, metal ions and surfactants on the fluorescence intensity has been studied. The proposed method allows the determination of 5-100 ng ml(-1) of velnacrine in aqueous solution containing sodium acetate buffer (pH 5.6; 0.04 M) with lambda(excitation) 242 nm and lambda(emission) 359 nm. The detection and quantitation limits were 1.7 and 4.5 ng ml(-1) respectively. The method was successfully applied to the determination of velnacrine in human serum and urine.