Concentrations of plasma histamine and serum neutrophil chemotactic factor (NCF) were measured in seven atopic asthmatics who developed exercise-induced asthma (EIA) after a treadmill task. The results were compared with those obtained after inhalation of specific antigen or methacholine. Plasma histamine concentrations were measured with a novel double-isotope radiometric assay, and NCF was identified by its elution in the void volume fractions of Sephadex G-200 and as a single peak of activity at approximately 0.20 molar NaCl after anion exchange chromatography on diethylaminoethyl-Sephacel (pH 7.8). After exercise or antigen challenge, the time courses of appearance of both mediators were virtually identical and accompanied the increase in airways obstruction. There was a statistically significant correlation between the concentrations of histamine or NCF and the magnitude of airflow obstruction after exercise and antigen challenge. This suggested that there may be a direct association between mediator release and EIA or antigen-induced bronchoconstriction. In contrast, there were no significant elevations in circulating histamine and NCF after inhalation of methacholine, at concentrations giving a fall in FEV1 comparable to that induced by exercise or antigen. The prior administration of cromolyn to three asthmatics inhibited both their EIA and the release of histamine and NCF. When four asthmatics were exercised for periods of 1, 3, and 6 min, the release of NCF and fall in peak expiratory flow rate were directly related to the duration of the exercise. The rise of NCF activity in subjects with EIA was fivefold greater than that observed in asthmatics who did not experience airways obstruction when subjected to the same exercise task. These results provide further evidence that mediators of hypersensitivity are released during EIA.