A method for determining the molecular species composition of phosphatidylcholine in biological samples by reversed-phase high-performance liquid chromatography with dual-wavelength ultraviolet detection is described. The optimum compromise between analysis time and chromatographic resolution under isocratic and isothermal conditions (0.8 ml/min and 32 degrees C, respectively) was obtained with the mobile phase methanol-ethanol (6:4, v/v) containing 20 mM choline chloride-water-acetonitrile (90:7:3, v/v/v). The problems of quantification at 205 nm, due to large differences in the detector response with the degree of unsaturation, were resolved by using the appropriate calibration factors chosen with the ratio of absorbances at 205 and 215 nm. The proposed procedure gave results in good agreement with fatty acid composition in samples of rat bile, liver, liver mitochondria and microsomes determined by gas-liquid chromatography.