Previous studies in vivo or in isolated airway preparations have suggested that eosinophil-derived polycationic proteins enhance airway smooth muscle tone in an epithelium-dependent manner. We assessed the direct effects of activated human eosinophil supernatant, major basic protein (MBP), and polycationic polypeptides on basal and agonist-stimulated intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in cultured bovine tracheal smooth muscle (TSM) cells. A 1-h incubation of myocytes with activated eosinophil buffer resulted in a doubling of basal [Ca²⁺]_i and increased responsivity to histamine compared with myocytes that were exposed to sham-activated eosinophil buffer. In addition, concentration-dependent acute transient increases and subsequent 1-h sustained elevations of basal [Ca²⁺]_i were observed immediately after addition of MBP and model polycationic proteins. Finally, both peak and plateau [Ca²⁺]_i responses to bradykinin addition were augmented significantly in cultured myocytes that had been exposed to low concentrations of MBP or model polycationic proteins but were inhibited at greater concentrations. This elevated [Ca²⁺]_i to polycationic proteins was manifest in epithelium-denuded bovine TSM strips as concentration-dependent increased basal tone. We conclude that activated eosinophil supernatant, MBP, and other polycationic proteins have a direct effect on both basal and subsequent agonist-elicited Ca²⁺ mobilization in cultured TSM cells; TSM strips in vitro demonstrated, respectively, augmented and diminished responses to the contractile agonist acetylcholine. It is possible that alteration in myocyte Ca²⁺ mobilization induced by these substances may influence clinical states of altered airway tone, such as asthma.