Increased resistance of airways or blood vessels within the lung is associated with asthma or pulmonary hypertension and results from contraction of smooth muscle cells (SMCs). To study the mechanisms regulating these contractions, we developed a mouse lung slice preparation containing bronchioles and arterioles and used phase-contrast and confocal microscopy to correlate the contractile responses with changes in [Ca²⁺]_i of the SMCs. The airways are the focus of this study. The agonists, 5-hydroxytrypamine (5-HT) and acetylcholine (ACH) induced a concentration-dependent contraction of the airways. High concentrations of KCl induced twitching of the airway SMCs but had little effect on airway size. 5-HT and ACH induced asynchronous oscillations in [Ca²⁺]_i that propagated as Ca²⁺ waves within the airway SMCs. The frequency of the Ca²⁺ oscillations was dependent on the agonist concentration and correlated with the extent of sustained airway contraction. In the absence of extracellular Ca²⁺ or in the presence of Ni²⁺, the frequency of the Ca²⁺ oscillations declined and the airway relaxed. By contrast, KCl induced low frequency Ca²⁺ oscillations that were associated with SMC twitching. Each KCl-induced Ca²⁺ oscillation consisted of a large Ca²⁺ wave that was preceded by multiple localized Ca²⁺ transients. KCl-induced responses were resistant to neurotransmitter blockers but were abolished by Ni²⁺ or nifedipine and the absence of extracellular Ca²⁺. Caffeine abolished the contractile effects of 5-HT, ACH, and KCl. These results indicate that (a) 5-HT and ACH induce airway SMC contraction by initiating Ca²⁺ oscillations, (b) KCl induces Ca²⁺ transients and twitching by overloading and releasing Ca²⁺ from intracellular stores, (c) a sustained, Ni²⁺-sensitive, influx of Ca²⁺ mediates the refilling of stores to maintain Ca²⁺ oscillations and, in turn, SMC contraction, and (d) the magnitude of sustained airway SMC contraction is regulated by the frequency of Ca²⁺ oscillations.