Airway responses to inhalation challenge with methacholine in healthy nonasthmatic subjects were examined to determine whether attenuation of response occurred upon repeated challenge. In one experiment, progressively increasing tolerance to multiple-dose challenges was seen with 4 challenges at 4 h intervals. In another study, a smaller degree of tolerance was also seen when a single-dose challenge was conducted 3 times at 24 h intervals. In these studies, the doses of methacholine used to obtain responses in nonasthmatic subjects were higher than are required under conditions of clinical methacholine challenge as a diagnostic test for asthma. Consequently, these findings may be relevant only to investigations or epidemiologic studies in which serial methachloline challenges are performed at intervals of 24 h or less in nonasthmatic subjects requiring higher cumulative doses of methacholine than do asthmatics.