A reverse-phase high performance liquid chromatography (HPLC) method was developed which resolved isomers of lysophosphatidylcholine (LPC) differing in the location of the aliphatic chain (sn-1 or sn-2 position) and the position (Δ6 or Δ9) or geometric configuration (cis or trans) of the olefin group in monounsaturated species. LPC isomers containing an acyl substituent at the sn-2 position eluted before their 1-acyl-sn-glycero-3-phosphocholine (1-acyl LPC) counterparts. The retention times of both the sn-1 and sn-2 isomers of monounsaturated species increased in the order Δ9-cis < Δ9-trans < Δ6-cis. The integrated ultraviolet absorbance (203 nm) in binary mixtures of the Δ9-cis and Δ6-cis 2-acyl lysophospholipid isomers correlated with the lipid phosphorus content of corresponding column eluates (r-0.994). Thus, the present method will facilitate synthesis of isomerically pure diradylphospholipids by providing homogeneous lysophospholipid precursors and help simplify the quantitative analysis of unsaturated lysophospholipid species.