Serotonin and acetylcholine are bronchoconstrictors clinically relevant for airway diseases associated with airway hyperreactivity. Caveolins (cav-1, -2 and -3), which are binding partners for receptors and enzymes, are structural components of caveolae. The process of cav assembling into caveolae requires cytoplasmic adapter proteins (cavin-1 to -4). Here, we addressed the role of cav-1, cav-3, cavin-1 and -4 in airway smooth muscle (SM) constriction utilizing immunofluorescence, RT-PCR, electron microscopy, western blotting, co-immunoprecipitation and functional analysis (organ bath and videomicroscopy) in cav-1 and cav-3 deficient (KO) and wild-type mice. In mouse airways, cavin-1 and -4 interact with cav-1 and cav-3, respectively. Cav-1 expression was decreased in tracheal SM in cav-3-KO mice. Caveolar numbers were diminished in cav-1-KO but not in cav-3-KO tracheal SM. Muscarine-induced tracheal constriction was not altered in either KO-strain but was increased in intrapulmonary bronchi of cav-3-KO mice. However, serotonin-induced constriction was lost in the trachea of both KO-strains. Protein and mRNA for cavin-1, -4 and cav-1 and -3 were also detected in human intrapulmonary bronchi and primary SM cells. In conclusion, cav-1 and cav-3 are essential for serotonin-induced tracheal SM constriction pointing to an involvement of different serotonin receptors in the trachea and bronchi that differ in their coupling to caveolae. Cav-3 was previously found to interact with muscarinic receptor type-2 (m2AChR) in bronchial SM cells. Thus, depletion of cav-3 might cause a disturbance of m2AChR coupling to caveolae resulting in increased muscarine-induced bronchoconstriction. Funding: DFG (SFB-TR84)