This study used a beta-escin-permeabilized canine tracheal smooth muscle preparation to test the hypothesis that the volatile anesthetic halothane decreases myofilament Ca2+ sensitivity by inhibiting the membrane receptor-linked second messenger systems that regulate myofilament Ca2+ sensitivity and not by inhibiting Ca(2+)-calmodulin activation of the contractile proteins. Acetylcholine (ACh) caused a GTP-dependent increase in force at constant submaximal cytosolic Ca2+ concentration. ACh, guanosine-5'-O-(3-thiotriphosphate), and the protein kinase C agonist 12,13-phorbol dibutyrate each significantly decreased the concentration of free Ca2+ producing a half-maximal response from 0.77 +/- 0.09 microM (Ca2+ alone) to 0.16 +/- 0.01, 0.19 +/- 0.02, and 0.37 +/- 0.03 microM, respectively, demonstrating an increase in myofilament Ca2+ sensitivity. Halothane (0.92 +/- 0.12 mM) had no effect on the free Ca2+ concentration-response curves generated by Ca2+ alone. However, in the presence of 3 microM ACh plus 10 microM GTP to maximally activate muscarinic receptors, halothane significantly increased the EC50 for free Ca2+ from 0.17 +/- 0.01 microM to 0.38 +/- 0.03 microM. These findings suggest that halothane decreases myofilament Ca2+ sensitivity in beta-escin-permeabilized canine tracheal smooth muscle by inhibiting the membrane receptor-linked second messenger systems that regulate myofilament Ca2+ sensitivity.