Halothane is a potent bronchodilator that attenuates vagally mediated constriction of canine airways, in part by a direct action on the smooth muscle cell. In tracheal smooth muscle, acetylcholine (ACh) produces a transient peak increase in cytosolic ionized calcium concentration ([Ca2+](i)), which declines to a sustained plateau concentration that is higher than that of the unstimulated muscle, and a sustained increase in force. The transient peak [Ca2+](i) is caused primarily by Ca2+ release from the sarcoplasmic reticulum (SR), whereas the plateau [Ca2+](i) is caused primarily by influx of extracellular Ca2+ across the plasma membrane. This study was conducted to investigate the effects of halothane on the 1) transient peak [Ca2+](i) during force development (i.e., developed force) and 2) plateau [Ca2+](i) during force maintenance (i.e., sustained force) induced by ACh in isolated strips of canine tracheal smooth muscle. Changes in [Ca2+](i) were measured with the photoprotein, aequorin. In muscle strips contracted after incubation with 1.1 (n = 5) or 1.5 (n = 5) minimum alveolar concentration (MAC) halothane, halothane significantly attenuated the transient peak aequorin luminescence (AL) and developed force and significantly prolonged the time necessary to reach peak AL and developed force; these effects were not dose dependent. In muscle strips (n = 3) contracted with ACh, addition of halothane caused a reduction in sustained force but no decrease in plateau AL. These results suggest that in canine tracheal smooth muscle in vitro, clinically relevant concentrations of halothane attenuates the increase in both [Ca2+](i) and developed force induced by ACh, possibly by inhibiting Ca2+ release from the SR, whereas during sustained force halothane 1) relaxes the tracheal smooth muscle by cellular mechanisms independent of [Ca2+](i), possibly by interfering with Ca2+- and calmodulin-mediated cellular events or, alternatively, the molecular processes that modulate the sensitivity of the contractile elements to [Ca2+](i), and 2) does not interfere with Ca2+ influx across the plasma membrane.