Methacholine chloride is a powerful cholinergic bronchoconstrictor agent used during bronchial airway hyper-responsiveness diagnosis. Methacholine is susceptible to hydrolysis in aqueous solutions in acetic acid and beta-methylcholine. In the present work, kinetics of hydrolysis with different solvents (water and phosphate-buffered saline (PBS) pH 7.4) at different temperatures have been studied using a newly developed high-performance liquid chromatography. At 4 degrees C, kinetic determination of hydrolysis in methacholine chloride solutions (50 mg/ml) shows no hydrolysis in either aqueous or phosphate-buffered solutions over a 40-day period. At 30 degrees C, concentration of unbuffered methacholine chloride solutions remained unchanged, but buffered methacholine chloride solutions have degradation up to 5.5% over a 40-day period. At 40 degrees C, concentration of unbuffered methacholine chloride has degradation up to 5% and buffered methacholine chloride solutions have degradation up to 10% over a 40-day period. Methacholine chloride solutions are susceptibly to be used in hospital pharmacy at different concentrations. We have studied pH and osmolality for methacholine solutions prepared with different diluents potentially used in hospital pharmacies, i.e. deionized water, 0.9% NaCl and PBS pH 7.4. We have demonstrated that methacholine solutions prepared with deionized water at 50 mg/ml and diluted with PBS pH 7.4 from 5 to 40 mg/ml are isoosmotic and potentially available for inhalation tests to measure non-specific bronchial hyper-responsiveness.