A simple HPLC method for the simultaneous analysis of phosphatidylcholine (PC), 1-acyl lysophosphatidylcholine (1-acyl LPC), and 2-acyl lysophosphatidylcholine (2-acyl LPC) with refractive index detection is described. The separation of these three compounds was achieved on a Waters (Milford, MA) Spherisorb amino phase column using a mixture of ethanol and an aqueous oxalic acid solution as eluent. The optimal mixture of ethanol to oxalic acid solution was 92∶8 (vol/vol). PC and the two regioisomers of LPC eluted within 15 min. The calibration curves were linear in a concentration range from 0.05 to 2.5 mM. Natural PC or LPC eluted in a single peak, despite the diversity in the fatty acid composition. There was no rearrangement between 1-acyl LPC and 2-acyl LPC during analysis or storage in ethanol within 23 h. This method is thus especially suitable for studying reactions on PC and acyl migration in LPC.