To explain that bronchial smooth muscle undergoes sustained agonist-induced contractions in a Ca2+-free medium, we hypothesized that caveolae in the plasma membrane (PM) contain protected Ca2+. We isolated caveolae from canine tracheal smooth muscle by detergent treatment of PM-derived microsomes. Detergent-resistant membranes were enriched in caveolin-1, a specific marker for caveolae as well as for L-type Ca2+ channels and Ca2+ binding proteins (calsequestrin and calreticulin) as determined by Western blotting. Also, the PM Ca2+ pump was present but not connexin 43 (a noncaveolae PM protein), the sarcoplasmic reticulum (SR) Ca2+ pump, or the type 1 inositol 1,4,5-trisphosphate receptor, supporting the idea that SR-derived membranes were not present. Antibodies to caveolin coimmunoprecipitated caveolin with calsequestrin or calreticulin. Thus some of the cellular calsequestrin and calreticulin associated with caveolin on the cytoplasmic face of each caveola. Immunohistochemistry of tracheal smooth muscle crysosections confirmed the localization of caveolin and the PM Ca2+ pump to the cell periphery, whereas the SR Ca2+ pump was located deeper in the cell. The presence of L-type Ca2+ channels, the PM Ca2+ pump, and the Ca2+ bindng proteins calsequestrin and calreticulin in caveolin-enriched membranes supports caveola involvement in airway smooth muscle Ca2+ handling.