The alkenyl-acyl subclass of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (plasmalogens) are minor components of alveolar surfactant. Plasmalogens promote and stabilize hexagonal structures of phospholipids. In another study (W. R. Perkins, R. B. Dause, R. A. Parente, S. R. Michey, K. C. Neuman, S. M. Gruner, T. F. Taraschi, and A. S. Janoff. Science 273: 330-332, 1996), it was shown that polymorphic phase behavior may have an important role in the effective functioning of pulmonary surfactant. Therefore, we hypothesized that surface properties of phospholipid mixtures that contain plasmalogens are superior to plasmalogen-free mixtures. The effect of plasmalogens on surface tension of surfactant-like phospholipid mixtures (70 mol% dipalmitoyl phosphatidylcholine, 10 mol% phosphatidylglycerol, and 20 mol% PtdEtn) was measured. Using the pulsating bubble surfactometer, we show that an increasing amount of ethanolamine plasmalogens [plasmenylethanolamine (PlsEtn)] results in reduction of surface tension (0 mol% PlsEtn 44.7 ± 1.7, 2 mol% 33.5 ± 1.7, 4 mol% 36 ± 3.1, 6 mol% 26.2 ± 2.9, and 8 mol% 22.2 ± 0.3 mN/m). By means of the captive bubble surfactometer, minimal surface tension reached with 8 mol% PlsEtn was even lower (3.8 ± 0.7 mN/m). With regard to morphological studies (B. Fringes, K. Gorgas, and A. Reith. Eur. J. Cell Biol. 46: 136-143, 1988), clofibrate treatment of rats might increase the plasmalogen content of alveolar surfactant. However, in the present study, we could not show that synthesis and secretion of plasmalogens are affected by clofibrate treatment.