Methacholine inhalation challenge has been shown to be an extremely useful diagnostic test. The purpose of this study was to document the reproducibility of methacholine inhalation challenge used in our clinic. In addition, we also examined the output of the delivery system, the stability of four-month-old methacholine solution, and the cumulative effect of methacholine. To document reproducibility, two identical challenges were performed in each of 19 asthmatic children. The influence of the previous doses of methacholine on bronchial response was examined by performing a third challenge with a single dose of methacholine in ten of these children. The remaining nine children were also tested for the third time with four-month-old methacholine solution to examine the stability of that solution. The output of the delivery system was assessed by measuring the change in weight of the nebulizer. Responses to methacholine were highly reproducible within one doubling dose interval. There was a small but significant cumulative effect of methacholine. Comparable results were obtained with newly prepared methacholine solution and four-month-old solution. The variability of the output of the same nebuliser was less than that of different nebulisers of the same model. The major clinical implication of our results is that our methacholine inhalation challenge procedure is standardized. This encourages more widespread use of this important diagnostic test for demonstration of airway hyper-responsiveness.