Methacholine (MCh) challenge testing is often incorporated into clinical studies prior to performing bronchoscopy as a measure of bronchial hyperresponsiveness (BHR). However, the effect of methacholine on many aspects of bronchoalveolar lavage (BAL) fluid cell count and function have not been fully evaluated. Ten patients with asthma, maintained on inhaled beta 2-agonists, were studied. Each subject underwent two bronchoscopies in a random order, one preceded by methacholine challenge within 30 min of the BAL. The investigators were blinded to the regimen. Several markers of BAL fluid cell number and function were studied: cell count and differential histamine, eosinophil products, including eosinophil cationic protein and Charcot-Leyden crystal protein, macrophage production of thromboxane B2 and leukotriene B4, neutrophil lysozyme and lactoferrin, and lymphocyte typing and activation markers measured via flow cytometry. No significant differences were noted in any of these markers of cell number or function which could be ascribed to methacholine challenge. Thus, methacholine challenge does not appear to affect these markers of cell number and function. These findings indicate that a methacholine challenge can be used as a measure of bronchial hyperresponsiveness within 30 min prior to bronchoscopy without altering bronchoalveolar lavage fluid characteristics.