Methacholine, provided by industrial sources, has traditionally been used in studies of airways responsiveness. In 1986, a Food and Drug Administration approved formulation of methacholine (Provocholine) was released and replaced industrial methacholine in many pulmonary laboratories. To determine whether methacholine and Provocholine cause an equivalent degree of bronchoconstriction, a double blind, cross-over clinical trial was undertaken. After randomization, 19 medicine residents and respiratory therapists each performed methacholine challenge testing using either methacholine or Provocholine. Forty-eight hours later, each participant returned for repeat challenge testing with the alternate agent. The log of the dose-response slope (logslope) was calculated for each test. The mean logslope with methacholine (-0.15 +/- 1.84) and with Provocholine (-0.26 +/- 1.57) did not differ (paired Student's t test, p = 0.64). Further, excellent agreement was found between each subject's logslope with methacholine and with Provocholine (intraclass correlation coefficient rI = 0.82). Proton beam nuclear magnetic resonance revealed no structural differences between the two compounds. These findings suggest that methacholine from industrial sources and Provocholine are clinically and structurally similar and that the two agents may be used interchangeably in nonspecific bronchial provocation testing.