Phospholipid parameters - lecithin/sphingomyelin (L/S) ratio and palmitic acid/stearic acid (P/S) ratio - of gastric (GA), pharyngeal (PA), and tracheal aspirates (TA) have often been used for estimation of lung maturation. However, it was not known whether values from different sources can be alternatively used. The study was performed to test whether P/S and L/S ratios in GA, PA, and TA are comparable. 11 GA, 30 PA, and 10 TA were obtained from infants within 10 min after birth. All infants were healthy, vaginally delivered with no respiratory distress, and had a gestational age ≤ 34 weeks. Patterns of fatty acids were determined by gas chromatography and the L/S ratio by high-performance liquid chromatography. We found lower L/S ratios in GA (16.5 ± 5.2) when compared with PA (39 ± 16) or TA (41 ± 15; p < 0.01); there were no differences between PA and TA. The P/S ratio was in the same range in PA and TA, but lower in GA. As compared with TA and PA, in GA the relative percentage of palmitic acid of all fatty acids was lower, the percentage of monounsaturated fatty acids, however, higher. We conclude that phospholipid parameters of lung maturation in PA and TA, but not in GA, can be alternatively used.