High-resolution 31P NMR spectroscopy was used to analyze the phospholipid composition of mammalian pulmonary surfactant from two different sources. Under conditions which considerably narrow the usually broad 31P phospholipid signals, solution-phase NMR spectra of these surfactant preparations unequivocally demonstrate that a phosphocholine plasmalogen (i.e., 1 -O-(1 ′-alkenyl) -2-acyl-sn-glycero-3-phosphocholine) exists as a major secondary component (∼4 mol %) in mammalian pulmonary surfactant. Phosphocholine (PC) plasmalogen was identified in preparations obtained from both adult cow lung surfactant extract as well as in ovine (lamb) fetal pulmonary liquid. PC plasmalogens have not previously been identified any mammalian pulmonary surfactant preparation. The amount of PC plasmalogen in these preparations occurs at fractional levels that are comparable to that of phosphoglycerol (PG), which previously had been thought of as the second-most common phospholipid class in pulmonary surfactant. The presence of PC plasmalogen in pulmonary surfactant may have important physiological ramifications and immediately suggests new directions for biochemical and biophysical investigations of pulmonary surfactant. © 1993, American Chemical Society. All rights reserved.