Formoterol, a long-acting ß₂-receptor agonist, is used to relieve bronchial constriction. However, formoterol is often a racemic formulation and contains both (R,R)- and (S,S)-enantiomers. Because the activity of each isomer is poorly defined, the mechanisms by which formoterol relaxes smooth muscle cells (SMC) of intrapulmonary airways are not well understood. Consequently, we compared the effects of (S,S)-, (R,R)- and racemic formoterol, as well as (R)-albuterol, on the contraction and Ca²⁺ signaling of airway SMCs in mouse lung slices with phase-contrast and confocal microscopy. Small airways were contracted with MCh and the associated SMCs displayed sustained Ca²⁺ oscillations and an increase in Ca²⁺ sensitivity. These contracted airways displayed a substantial concentration-dependent relaxation in response to (R,R)-formoterol. Racemic-formoterol had a similar potency as (R,R)-formoterol for relaxing airways. By contrast, (S,S)-formoterol only induced a small relaxation. In conjunction with relaxation, (R,R)- and racemic-formoterol stopped and decreased the MCh-induced Ca²⁺ oscillations and Ca²⁺ sensitivity of the SMCs, respectively whereas (S,S)-formoterol only decreased the Ca²⁺ sensitivity. In these studies, (R,R) and racemic-formoterol had a similar but much greater potency than (R)-albuterol for relaxing mice airways. This action was quickly initiated at high concentrations by decreasing the frequency of Ca²⁺ oscillations but was more usually mediated at lower concentrations by decreasing the Ca²⁺ sensitivity of the SMCs.