The ₂-agonists currently used as bronchodilators are racemic mixtures of R- and S-enantiomers. In the present study, we examined the effects of enantiomers of the ₂-agonists albuterol and formoterol on acetylcholine (ACh) release from equine trachealis parasympathetic nerves. ACh release was evoked by electrical field stimulation (20 V, 0.5 ms, 0.5 Hz) and measured by high-performance liquid chromatography coupled with electrochemical detection. We also tested the effects of enantiomers of albuterol and formoterol on equine tracheal smooth muscle (TSM) contraction in response to exogenous ACh. R- and RS-albuterol (10⁸ to 10⁵ M) and RR- and RR/SS-formoterol (10⁸ to 10⁵ M) augmented ACh release in a concentration-dependent manner. Beginning at 10⁶ M, SS-formoterol significantly increased ACh release, and at 10⁵ M, release increased by 71.9 ± 8.7% over baseline. This effect was only observed, however, when the prejunctional muscarinic autoinhibitory effect of ACh was prevented with atropine. Both the RR- and SS-formoterol-induced increases in ACh release were abolished by the ₂-antagonist ICI-118551 (3 × 10⁷ M). The effect of S-albuterol on ACh release was variable, and the mean increase induced by 10⁵ M was 30.8 ± 16.1% in the presence of atropine. In the muscle tension study, R- and RS-albuterol and RR- and RR/SS-formoterol (10⁸ to 10⁵ M) but not the S-enantiomers inhibited TSM contraction. Even though R-enantiomers augment ACh release, they potently inhibit TSM contraction. Because racemic ₂-agonists are bronchodilators on acute administration, the postjunctional spasmolytic effects of R-enantiomers predominate over the spasmogenic effect evoked via increased ACh release. The S-enantiomers, in contrast, do not inhibit TSM contraction and therefore would not contribute to the observed bronchodilation of the racemate. The S-enantiomers do prejunctionally facilitate ACh release when prejunctional muscarinic autoreceptors are dysfunctional, suggesting a potentially deleterious effect.