A study was made of the responses of guinea pig tracheal muscle in vitro after sensitization by either subcutaneous or intraperitoneal administration of antigens. No difference was observed in the responses of strips from either group to cumulative or single doses of antigen. The responses of the tracheal muscle to antigen were analyzed as to magnitude (relative to maximal carbachol responses), time to peak contraction, and time to half decay. Pyrilamine, an H1-receptor antagonist, slowed the onset of contraction and decay. The latter result suggested a role for histamine in controlling the release of or the response to other mediators and the former was consistent with previous evidence that histamine release augments the initial response. An inhibitor of 5′-lipoxygenase and cyclooxygenase (5,8,11,14-eicosatetraynoic acid), indomethacin, and the slow-reacting substance of anaphylaxis (SRS-A) antagonist FPL-55712 all shortened the time to half decay of the contraction, presumably by different mechanisms. The release and actions of leukotrienes may have been limited by eicosatetraynoic acid and FPL55712, respectively, but the action of indomethacin suggests a modultaing role for prostanoids in leukotriene action or release. Nordihydroguaiaretic acid both reduced the maximum response and accelerated decay of this response, suggesting that the maximum response depends upon leukotriene release. Cromoglycate, methysergide, and an inhibitor of thromboxane synthetase were without effect. Both tetrodotoxin and atropine delayed the time to peak contraction and tetrodotoxin also delayed the decay of the contraction. These results suggest that, in addition to the accepted occurrence of sequential histamine and leukotriene release, there may be a role for nerves and prostanoids in modulating mediator release but no role of thromboxanes or serotonin. Histamine may not only initiate the response but also may modulate the response to released leukotrienes. The lack of effect of cromoglycate is unexplained.