Phosphatidylcholine (PC) is the main phospholipid in lung surfactant and, more specifically, dipalmitoyl PC (PC16:0/16:0) is the major surface-active component. Several studies have tentatively shown that eustachian tube lavage fluid (ETLF) contains surface-active material. The aim of the present study was to determine, using electrospray ionization mass spectrometry, whether the phospholipid molecular species composition of ETLF is similar to that of lung surfactant. PC was the main component of both ETLF and bronchoalveolar lavage fluid (BALF). The concentration of phosphatidylethanolamine was higher and phosphatidylglycerol was undetectable in ETLF compared with BALF. The molecular species composition of PC in ETLF was notably different from that of BALF, palmitoyloleoyl PC being the major component. Importantly, given its predominance in BALF PC, the concentration of PC16:0/16:0 was low in ETLF. As expected on the basis of this molecular species composition of PC, ETLF did not generate low surface tension values under dynamic compression in a pulsating bubble surfactometer. We conclude that the surfactant in ET is different from lung surfactant, and that low surface tension is not a major determinant of ETLF function.