The ₂-adrenergic agonist, albuterol, is used as a bronchodilator by asthma patients and consists of a racemic mixture of (R)- and (S)-albuterol. However, the action of the individual enantiomers is poorly understood. Consequently, we investigated the effects of (R)-, (S)- and racemic-albuterol on airway smooth muscle cell (SMC) contraction and Ca²⁺ signaling in mouse lung slices with phase-contrast and confocal microscopy. (R)-albuterol relaxed airways contracted with methacholine (MCh) in a dose-dependent manner. By contrast, (S)-albuterol had no effect on airways. (R)-albuterol had a greater relaxant effect than a double concentration of racemic albuterol. Because MCh-induced contraction of airway SMCs is mediated by Ca²⁺ oscillations and an increase in Ca²⁺ sensitivity, the effects of albuterol on these responses were examined. Both (R)- and racemic albuterol decreased the frequency of the MCh-induced Ca²⁺ oscillations by a similar amount. However, (R)-albuterol was more effective than racemic albuterol in decreasing the Ca²⁺ sensitivity of the airway SMCs in "model" lung slices with a clamped [Ca²⁺]_i. In contrast, (S)-albuterol had no effect on the Ca²⁺ oscillations or the Ca²⁺ sensitivity. In conclusion, (R)-albuterol consistently induced a greater airway relaxation than racemic albuterol and (S)-albuterol appears to be responsible for this reduced efficacy.