The mechanism of the direct relaxing effect of halothane on airway smooth muscle may involve a decrease in 1) cytosolic calcium concentration ([Ca2+](i)) and/or 2) the force produced for a given [Ca2+](i) (i.e., the 'sensitivity' of the myofibrillar contractile system to Ca2+). This study was conducted to test the hypothesis that halothane reduces the sensitivity of the myofibrillar contractile system to Ca2+ during muscarinic receptor stimulation of canine tracheal smooth muscle. Isolated smooth muscle strips were mounted in a photometric superfusion system, stretched in their optimal length for force development, and loaded with the fluorescent Ca2+ indicator, fura 2, for simultaneous recording of fura 2 fluorescence and isometric force. Emission fluorescence intensities due to excitation at 340 (F340)- and 380 (F380)-nm wavelengths were measured and F340/F380 was used as an index of [Ca2+](i). After superfusion with Ca2+-free physiological salt solution (PSS) containing 1 or 100 μM acetylcholine (ACh), two consecutive cumulative concentration-response curves to CaCl2 (0.01-2.4 mM) were generated for each strip; one curve was generated in the presence of halothane. In strips stimulated with 1 (n = 6) or 100 (n = 6) μM ACh, the cumulative addition of CaCl2 to the Ca2+-free PSS caused concentration-dependent increases in both F340/F380 and force. In strips stimulated with 1 μM ACh, 2.4 ± 0.3% halothane proportionally attenuated increases in both F340/F380 and force. In strips stimulated with 100 μM ACh, 2.5 ± 0.4% halothane attenuated increases in force, but not increases in F340/F380, thereby causing a rightward shift of the F340/F380-force relationship. These data suggest that the cellular mechanism for the direct relaxing effect of halothane on canine tracheal smooth muscle depends on the intensity of muscarinic receptor stimulation. During stimulation with 1 μM ACh, halothane attenuated force primarily by reducing [Ca2+](i). In contrast, during stimulation with 100 μM ACh, halothane attenuated force primarily by reducing the sensitivity of the myofibrillar contractile system to Ca2+.