Using real-time confocal microscopy, we examined the dynamic intracellular Ca2+ concentration ([Ca2+]i) response of porcine tracheal smooth muscle (TSM) cells to acetylcholine (ACh). Exposure to ACh caused regenerative, propagating [Ca2+]i oscillations. The amplitude and fall time of the [Ca2+]i oscillations were inversely correlated to basal [Ca2+]i, whereas the frequency and rise time were directly correlated to basal [Ca2+]i. ACh-induced [Ca2+]i oscillations were initiated in the absence of extracellular Ca2+ and after membrane depolarization with KCl, suggesting that 1) [Ca2+]i oscillations primarily arise by release from internal stores such as the sarcoplasmic reticulum (SR), and 2) Ca2+ influx is necessary for maintenance of oscillations. Exposure to both caffeine and ryanodine inhibited ongoing ACh-induced [Ca2+]i oscillations, suggesting a role for caffeine-sensitive ryanodine receptor (RyR) SR Ca2+ channels. Inhibition of SR Ca2+ reuptake by thapsigargin increased basal [Ca2+]i and decreased [Ca2+]i oscillation amplitude, suggesting that Ca2+ reuptake is also essential. The present results suggest that [Ca2+]i oscillations in porcine TSM cells involve repetitive Ca2+ release and reuptake from RyR channels, perhaps through a Ca2+ -induced Ca2+ release mechanism.