Ozone induces airway hyperresponsiveness, but there is controversy about effects of ozone on smooth muscle per se. We therefore investigated effects of in vivo ozone exposure on intracellular calcium mobilization in relation to tracheal smooth muscle contractility in the guinea pig in vitro. Guinea pigs underwent ozone exposure or sham exposure (3ppm, 2h). Then, a tracheal smooth muscle strip was mounted in an organ bath to record isometric tension. Effects of ozone exposure on acetylcholine-induced contraction of smooth muscle were as follows. Contraction was not altered in normal Krebs solution, but was increased in Ca²⁺-free solution in ozone-exposed animals. Decline of tension on repetitive application of acetylcholine in Ca²⁺-free solution was reduced, while the tension decline rate while acetylcholine was washed out with Ca²⁺-free solution was facilitated in ozone-exposed animals. Tension decline during the continuous administration of acetylcholine in Ca²⁺-free solution was slowed. Contraction occurred more quickly in Ca²⁺-free solution in ozone-exposed animals. Results suggest that ozone has a direct action on airway smooth muscle by changing Ca²⁺ mobilization; Ca²⁺ refilling via a Ca²⁺ pump and Ca²⁺ release via Ca²⁺ channels in the sarcoplasmic reticulum were increased, while Ca²⁺ extrusion via the plasma membrane Ca²⁺ pump was unchanged.