The molecular species of the major phospholipids from the marine sponges Parasperella psila and Microciona prolifera were studied using chemical hydrolysis, enzymatic degradation and capillary gas chromatography (GC), high performance liquid chromatography (HPLC), desorption chemical ionization (DCI), fast atom bombardment (FAB) combined with collisionally activated decomposition (CAD) mass spectrometry. Two new solvent systems were developed for the isolation of these species from the sponges. Our investigations indicated the existence of unusual symmetrical phospholipids as major components. 1,2-Di-(5 Z,9 Z)-5,9-hexacosadienoyl- sn-glycero-3-phosphoethanolamine was found in both organisms, while 1,2-di(5 Z,9 Z,19 Z)-5,9,19-hexacosatrienoyl- sn-glycero-3-phosphoethanolamine was present in M. prolifera. 1,2-Di-(4 Z,7 Z,10 Z,13 Z,16 Z,19 Z)4,7,10,13,16,19-docosahexaentoyl- sn-glycero-3-phosphocholine was the major molecular species in the PC fraction of M. prolifera.