Hydrogen sulfide (H 2 S) relaxes airway smooth muscle and it has been suggested to have a protective role during asthma and other obstructive lung diseases. However, the mechanism of H 2 S-induced airway relaxation is unknown. Smooth muscle cell (SMC) contraction depends on the phosphorylation state of the regulatory myosin light chain (rMLC) which is determined by the relative activities of the myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Changes in intracellular Ca 2+ concentration in SMCs regulate MLCK activity whereas Ca 2+ independent mechanisms regulate MLCP activity. In an accompanying abstract, we report that H 2 S inhibits intracellular Ca 2+ release through InsP 3 receptors and agonist-induced intracellular Ca 2+ oscillations in airway SMCs. Here we investigated the effect of H 2 S on rMLC phosphorylation in small intrapulmonary airways. We hypothesize that H 2 S-induced airway relaxation is accompanied by rMLC de-phosphorylation that results from a decrease in MLCK activity due to the inhibition of agonist-induced Ca 2+ oscillations. We used mouse lung slices in combination with phase-contrast- and confocal- video microscopy to measure contraction and Ca 2+ signaling in SMCs of small airways and used Western blot analysis to measure protein phosphorylation in airways micro-isolated from lung slices. Methacholine (MCh, 0.3 μM) induced strong and sustained airway contraction. This response was accompanied by the generation of intracellular Ca 2+ oscillations in airway SMCs that persisted in time and by a sustained increase in rMLC phosphorylation. Subsequent addition of 100 μM Na 2 S (a H 2 S donor) resulted in a strong and sustained airway relaxation. This relaxation was accompanied by a decrease in the frequency and amplitude of Ca 2+ oscillations and by rMLC de-phosphorylation. Washout of H 2 S in the continuous presence of MCh resulted in the increase in Ca 2+ oscillation frequency and amplitude, rMLC phosphorylation, and airway re-contraction. Our results suggest H 2 S induces relaxation of small intrapulmonary airways by inhibiting agonist-induced Ca 2+ oscillations which in turn induces rMLC de-phosphorylation in airway SMCs.