Reversed-Phase C₈ HPLC methods were developed for the separation of the molecular species of phosphatidylcholines (PC) and phosphatidylethanolamines (PE) using linear gradient of methanol-water containing ammonium hydroxide as silanol suppressor. The elution order of given PC and PE is inversely related to the polarity of its fatty acid constituents. For acyl chains with lower polarity, elution time increases as follows: ricinoleic acid < linolenic acid < myristic acid < palmitoleic acid < palmitelaidic acid  < arachidonic acid < linoleic acid < palmitic acid < oleic acid < elaidic acid < petroselinic acid < hexadecyl ether < stearic acid < arachidic acid. The molecular species of PC and PE incorporating various [¹⁴C]fatty acids, stearate, oleate, linoleate, linolenate, ricinoleate, palmitate and laurate, in castor and soybean microsomes were identified by matching the HPLC retention times of the standards using an absorbance detector (205 nm) and of the [¹⁴C]phospholipids incorporating [¹⁴C]fatty acids using a flow scintillation analyzer. When the standards were not available the identification was made by the HPLC elution characteristics of these lipid classes. The [¹⁴C]fatty acids were predominately incorporated at the sn-2 position of both PC and PE. Many relative retention times (RRT) of the molecular species of PC and PE are reported.