Fetal tracheal ligation increases lung growth in utero, making it potentially applicable for antenatal treatment of diaphragmatic hernia. This phenomenon has been ascribed to increased intratracheal pressure, which activates as yet unidentified pulmonary stretch receptors. The purpose of this study was to determine whether the composition of lung fluid has any effect on fetal lung development after tracheal obstruction. Six sets of fetal lamb twins underwent tracheal ligation with placement of intratracheal catheters at 122 days' gestation (term, 145 days). In group 1 (n = 6), tracheal fluid was aspirated daily, measured, and replaced with equal volumes of saline. Their respective twins (group 2, n = 6) had daily reinfusion of their own tracheal aspirates. Intratracheal pressure was recorded daily in both groups. Unobstructed fetal lambs (n = 7) were used as negative controls. Animals were killed on postoperative day 14 (136 days). Lungs were weighed, perfusion fixed at 25 cm H2O, and processed for standard morphometric analysis. Intratracheal pressure remained between 3 and 5 torr in both experimental groups throughout the entire postoperative period. In all 12 experimental fetuses, tracheal ligation resulted in an almost threefold increase in lung fluid volume by day 1; a slight decrease at a mean of 2.4 days; and a second surge from day 4 on. Lung fluid volume was significantly higher in group 2 than in group 1 at all measured time points (P < .05, Wilcoxon rank sum test) except on days 3, 4, and 8 (P = .06). Lung weight per body weight (LW/BW) at delivery was 0.045 +/- 0.008 in group 1, not significantly different from unobstructed controls (0.038 +/- 0.006). LW/BW in group 2 was 0.055 +/- 0.010, significantly larger than either group 1 or control (P < .05, single factor analysis of variance). Air space fraction was comparable between the three groups. Alveolar numerical density was significantly lower in groups 1 and 2 than in unobstructed controls (P < 0.05). Replacement of tracheal fluid with saline inhibits the lung hypertrophy seen after tracheal ligation. This phenomenon therefore appears more dependent on tracheal fluid growth factors than on increased intratracheal pressure after obstruction. The immediate decrease in net lung fluid production after saline exchange suggests that these humoral factors play an important role in the initiation of lung cell proliferation.