Pulmonary surfactant (PS), a mixture of phospholipids and proteins secreted by alveolar type II cells, functions to reduce the surface tension in the lungs of all air-breathing vertebrates. Here we examine the control of PS during lung development in a homeothermic egg-laying vertebrate. In mammals, glucocorticoids and autonomic neurotransmitters contribute to the maturation of the surfactant system. We examined whether dexamethasone, epinephrine, and carbamylcholine hydrochloride (agonist for acetylcholine) increased the amount of PS secreted from cultured type II cells of the developing chicken lung. In particular, we wanted to establish whether dexamethasone would increase PS secretion through a process involving lung fibroblasts. We isolated and cocultured type II cells and lung fibroblasts from chickens after 16, 18, and 20 days of incubation and from hatchlings (day 21). Epinephrine stimulated phosphatidylcholine (PC) secretion at all stages, whereas dexamethasone stimulated secretion of PC at days 16 and 18. Carbamylcholine hydrochloride had no effect at any stage. This is the first study to establish the existence of similar cellular pathways regulating the development of surfactant in chickens and eutherian mammals, despite the vastly different birthing strategies and lung structure and function.