Background Halothane relaxes airway smooth muscle, in part, by decreasing the force produced for a given intracellular [Ca(2+)] (i.e., Ca(2+) sensitivity) during muscarinic stimulation, an effect produced by a decrease in regulatory myosin light-chain (rMLC) phosphorylation. The authors tested the hypothesis that halothane reduces rMLC phosphorylation during muscarinic stimulation at constant intracellular [Ca(2+)] by increasing smooth muscle protein phosphatase (SMPP) activity, without changing myosin light-chain kinase (MLCK) activity. Methods Enzyme activities were assayed in beta-escin permeabilized strips of canine tracheal smooth muscle. Under conditions of constant intracellular [Ca(2+)], the rate of rMLC phosphorylation was measured by Western blotting during inhibition of SMPP with microcystin-LR (to assay MLCK activity) or during inhibition of MLCK by wortmannin and adenosine triphosphate depletion (to assay SMPP activity). The effect of halothane (0.8 mm) on enzyme activities and isometric force during stimulation with 0.6 microm Ca(2+) and 10 microm acetylcholine was determined. Results Halothane produced a 14 +/- 8% (mean +/- SD) decrease in isometric force by significantly reducing rMLC phosphorylation (from 32 +/- 9% to 28 +/- 9%). Halothane had no significant effect on any parameter of a monoexponential relation fit to the data for the MLCK activity assay. In contrast, halothane significantly decreased the half-time for rMLC dephosphorylation in the SMPP activity assay (from 0.74 +/- 0.28 min to 0.44 +/- 0.10 min), indicating that it increased SMPP activity. Conclusions Halothane decreases Ca(2+) sensitivity and rMLC phosphorylation in airway smooth muscle during muscarinic receptor stimulation by increasing SMPP activity, without affecting MLCK, probably by disrupting receptor G-protein signaling pathways that inhibit SMPP.