Previous studies have documented the presence of a sodium, potassium adenosine triphosphatase (Na+,K+ ATPase) enzyme inhibitor on platelet membranes and in the plasma of patients with allergy, many of whom historically had airway hyperreactivity (AHR). The purpose of this study was to investigate the relationship between methacholine AHR and Na+,K+ ATPase enzyme inhibition. In the first experiment, 47 adult subjects (13 allergic, 5 potentially allergic, 12 asthmatic, and 17 control subjects) were tested for platelet membrane Na+,K+ ATPase inhibition and AHR. Area under the methacholine dose-response curve (AUC) was expressed as percent baseline FEV1 x log concentration of methacholine (log [mg/ml]) and plotted as a function of the difference in postfreezing and prefreezing platelet membrane Na+,K+ ATPase activities (reflective of membrane-bound inhibitor), which was expressed as nanomoles per microgram of protein per minute (nmol/microg protein/min). A significant (r = -0.44, p < 0.005) negative correlation between the two was detected, such that high levels of AHR (low AUC) were associated with high levels of membrane-bound inhibitor. To test for a causal relationship between the two, the ability of a Na+,K+ ATPase inhibitor to directly influence the level of AHR was determined in a second experiment. Eight allergic and 10 control subjects were administered AHR tests on 2 different days, immediately after inhalation of either nebulized ouabain (1 mg) or placebo in a double-blind fashion. Ouabain versus placebo inhalation decreased the PC20 in four of the patients with allergy. Additionally, ouabain increased methacholine AHR in patients with allergy, as manifested by a lower AUC in seven of the eight patients. In contrast, the mean AUC for the ouabain versus placebo prechallenges did not change significantly in the control group. Finally, a positive correlation was demonstrated between the levels of platelet membrane Na+,K+ ATPase inhibition and bronchial responsiveness to ouabain (r = 0.49, p < 0.05). These results provide both correlative and mechanistic evidence for a causal relationship between Na+,K+ ATPase enzyme inhibition and AHR.