Hyperreactive responses of asthmatics to bronchoconstrictor agonists are caused by mediators produced by a variety of cell types within the airways. Nerves, mast cells, eosinophils, airway epithelia and airway smooth muscle cells probably all interact to increase bronchomotor tone in response to challenge with allergenic stimuli. In addition to important cell-cell interactions, there may be a significant postjunctional component of airway hyperreactivity in which mediators interact at the level of airway smooth muscle cells. To test this hypothesis, I investigated synergistic interactions of methacholine (MCH), histamine and serotonin using intact and chemically skinned canine tracheal smooth muscle. A threshold concentration of histamine (100 nM) significantly increased force when it was added 20 min after or 10 min before stimulation with low concentrations of MCH (1-10 nM). A threshold concentration of serotonin (10 nM) also significantly increased contractions induced by MCH. Serotonin (10 nM) added during a steady-state contraction induced by 6 or 10 nM MCH increased force and increased fura-2 fluorescence, which suggests that force increases in part because myoplasmic Ca++ increases. I also tested the hypothesis that mechanisms beyond the initial steps of signal transduction contribute to synergism by permeabilizing tracheal smooth muscle fibers with staphylococcal alpha-toxin. Histamine (30 microM), MCH (1 microM), GTP-gamma-S (100 microM) and phorbol dibutyrate (1 microM) all potentiated contractions induced by 1 microM Ca++ in skinned fibers. These data suggest that postjunctional components of airway hyperreactivity may be a combination of synergistic effects of agonists occurring at the level of excitation-contraction coupling as well as sensitization of contractile proteins to Ca++.