Background Halothane directly relaxes airway smooth muscle partly by decreasing the Ca2+ sensitivity. In smooth muscle, receptor stimulation is thought to increase Ca2+ sensitivity via a cascade of heterotrimeric and small monomeric guanine nucleotide-binding proteins (G-proteins). Whether this model is applicable in the airway and where halothane acts in this pathway were investigated. Methods A beta-escin-permeabilized canine tracheal smooth muscle preparation was used. Exoenzyme C3 of Clostridium botulinum, which inactivates Rho monomeric G-proteins, was used to evaluate the involvement of this protein in the Ca2+ sensitization pathway. The effects of halothane on different stimulants acting at different levels of signal transduction were compared: acetylcholine on the muscarinic receptor, aluminum fluoride (AIF4-) on heterotrimeric G-proteins, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) on all G-proteins. Results Exoenzyme C3 equally attenuated acetylcholine- and AIF4--induced Ca2+ sensitization, suggesting that these pathways are both mediated by Rho. Halothane applied before stimulation equally attenuated acetylcholine- and AIF4--induced Ca2+ sensitization. However, when added after Ca2+ sensitization was established, the effect of halothane was greater during Ca2+ sensitization induced by acetylcholine compared with AIF4-, which, along with the previous result, suggests that halothane may interfere with dissociation of heterotrimeric G-proteins. Halothane applied during GTPgammaS-induced Ca2+ sensitization had no significant effect on force, suggesting that halothane has no effect downstream from monomeric G-proteins. Conclusion Halothane inhibits increases in Ca2+ sensitivity of canine tracheal smooth muscle primarily by interfering with the activation of heterotrimeric G-proteins, probably by inhibiting their dissociation.