A variety of clinical and experimental evidence indicates that surfactant may be important in the pathogenesis and treatment of asthma. The purpose of this study was to determine the pharmacologic effect of pulmonary surfactant and its major lipid and protein constituents on bronchial smooth muscle. First-generation bronchi from male Sprague-Dawley rats were contracted with methacholine and exposed to two kinds of surfactant: whole rat surfactant and two bovine surfactant extracts in clinical use. The latter lack the hydrophilic surfactant-associated proteins (SP)-A and SP-D. All the surfactants relaxed the rat bronchi in a concentration-dependent manner; however, whole rat surfactant was more potent than the bovine extracts. Both surfactant lipids and SP-A contributed to the bronchial relaxation. The relaxation response produced by the highest concentration (0.5 mg/ml) of whole rat surfactant was equivalent to that caused by substance P (5 μM) and approximately half of that caused by 1 μM isoproterenol. The relaxation response was epithelium-dependent and blocked by indomethacin but not by N-ω-nitro-L-arginine methyl ester. We conclude that surfactant can relax airway smooth muscle directly via a prostanoid-mediated, epithelium-dependent process that does not involve nitric oxide synthase.