The enantiomeric resolution of six beta2-agonists including bambuterol, clenbuterol, clenproperol, fumoterol, mabuterol and terbutaline, was studied on three macrocyclic antibiotic chiral stationary phases (CSPs): Chirobiotic V, T and R. By varying the mobile phase compositions for different CSPs, all the six compounds were successfully separated on Chirobiotic V and T CSPs. The thermodynamic parameter variation was calculated. The chiral recognition mechanism was discussed, the ionic interaction was confirmed to be the most dramatic interaction responsible for the chiral recognition. No enantiosepartion for all the beta2-agonists was found on Chirobiotic R CSP.