To study the effect of lung expansion at birth on surfactant secretion, we delivered by hysterotomy 11 litters of rabbit pups at 30 days of gestation and divided them into three groups that were killed: 1) after 30 min air-breathing, 2) after 30 min nitrogen-breathing, and 3) after 30 min tracheal occlusion. Each group was compared to a littermate group killed at birth. Groups 2) and 3) continued respiratory efforts for 30 min despite progressive asphyxia. Six additional litters were pretreated with atropine; at delivery one-half of each litter was killed, the remaining pups were subjected to 30 min of hypoxic gas breathing. After sacrifice, alveolar surfactant was recovered by saline lavage and estimated quantitatively on a surface-tension balance. Surfactant concentration at birth was 1.40 +/- 0.22 mg/g dry lung and increased to 1.86 +/- 0.19 (+/- SEM) after 30 min air-breathing (P less than 0.01). Also, surfactant was increased in the nitrogen-breathing pups (from 1.61 +/- 0.35 in littermate controls to 2.41 +/- 0.58; P less than 0.03), but not to a significant degree in the occluded group (1.34 +/- 0.33 vs. 1.41 +/- 0.28), or the atropine pretreated breathing pups (1.77 +/- 0.29 vs. 1.89 +/- 0.25).