An isocratic, rapid method for separation of phospho- and lysophospholipids by high-performance liquid chromatography employing acetonitrile, methanol and water as the mobile phase is described. Separation is achieved on a silica based cation-exchange column with individual components monitored directly by UV absorbance at 203 nm. In solutions of phospho- and lysophospholipid standards and in extracts of rabbit myocardium, recovery of individual components exceeds 95%. With the exception of phosphatidyl serine and phosphatidyl ethanolamine that could not be resolved other major phospho- and lysophospholipid constituents found in extracts of cell membranes are separated completely within 40 min. Retention time for selected moieties can be shortened even further with flow programming.