Surfaces of lungs are covered by the surfactant, an aqueous mixture of different phospholipids (PL) and proteins. Although the surfactant represents a relatively simple mixture of only a few PL (primarily phosphatidylcholine (PC) and phosphatidylglycerol (PG)), reliable methods of routine lipid analysis of the surfactant are still lacking. It will be shown that matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS) represents a suitable technique for the differentiation of the apolar components of the surfactant of different species. Samples of man and minipig are used in this study since both are known to vary in their PL composition. PL of surfactant were separated by thin-layer chromatography (TLC) and the obtained subfractions subjected to MALDI-TOF MS analysis in order to monitor the presence of even minor PL species. It will be shown that besides PG and PC, also phosphatidylethanolamine, -inositol and sphingomyelin can be detected in surfactant of man, whereas only sphingomyelin could be detected in the minipig sample.