Conditions are described for the efficient resolution by reversed-phase high-performance liquid chromatography on octadecylsilica stationary phases of individual molecular species of phosphatidylcholine on the basis of their fatty acyl composition. The effects are described of varying stationary phase carbon loading, mobile phase polarity and column temperature. Eluted peaks were detected by a sensitive post-column fluorescence system using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. This detection system permitted direct quantitation of phosphatidylcholine species irrespective of the degree of acyl unsaturation and eliminated the need for elaborate pre-column derivatization. The techniques were applied to the analysis of phosphatidylcholine isolated from rat liver and lung.