Halothane directly relaxes airway smooth muscle. To determine the direct inhibitory mechanisms of halothane on canine tracheal smooth muscle contraction, the effects of this anesthetic on the levels of several intracellular second messengers were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i), Ca2+/phospholipid-dependent protein kinase (PKC) translocation, and intracellular cyclic adenosine monophosphate concentration ([cAMP]i). When carbachol (1 μgM) was used to increase [Ca2+]i to the same concentration as that induced by high-K+ (72.7 mM), the carbachol-induced contraction was more than twice as great, indicating that carbachol enhances the sensitivity of contractile elements to Ca2+ or activates a Ca2+-independent mechanism. Similarly, 12-deoxyphorbol 13-isobutylate, a potent PKC activator, markedly potentiated high-K+-induced muscle contraction without an increase of [Ca2+]i. The addition of halothane (0.33, 0.75, 1.15, and 1.47 mM) decreased [Ca2+]i and the muscle tension induced by carbachol. However, the decrease of muscle tension was more marked than that of [Ca2+]i at the higher concentrations. Although [Ca2+]i in the presence of verapamil and carbachol was not affected by halothane, the anesthetic markedly decreased muscle force by decreasing the “Ca2+ sensitization‘’ or the Ca2+-independent enhancement of tension observed with carbachol. Halothane (0.75 and 1.47 mM) significantly released the membrane-associated PKC to cytosol, which decreases PKC activity. [cAMP]i of the smooth muscle stimulated by carbachol was moderately but significantly increased by halothane. However, when equivalent relaxation was induced with forskolin, which acts via adenylate cyclase activation, a much higher [cAMP]i was observed, which suggests that halothane acts via an additional pathway. In conclusion, halothane-inhibits tracheal smooth muscle contraction, in part by decreasing [Ca2+]i, an effect that may be mediated partially by an increase in [cAMP]i. Another mechanism appears to be involved that is independent of Ca2+; this mechanism appears to depress the Ca2+ sensitization mediated by PKC.