The bioactive sphingolipid sphingosine‐1‐phosphate (S1P) that is increased in airways of asthmatic subjects markedly induced contraction of human airway smooth muscle (HASM) cells embedded in collagen matrices in a G_i‐independent manner. Dihydro‐S1P, which binds to S1P receptors, also stimulated contractility. S1P induced formation of stress fibers, contraction of individual HASM cells, and stimulated myosin light chain phosphorylation, which was inhibited by the Rho‐associated kinase inhibitor Y‐27632. S1P‐stimulated HASM cell contractility was independent of the ERK1/2 and PKC signaling pathways, important regulators of airway smooth muscle contraction. However, removal of extracellular calcium completely blocked S1P‐mediated contraction and Y‐27632 reduced it. S1P also induced calcium mobilization that was not desensitized by repeated additions. Pretreatment with thapsigargin to deplete InsP₃‐sensitive calcium stores partially blocked increases in [Ca²⁺]_i induced by S1P, yet did not inhibit S1P‐stimulated contraction. In sharp contrast, the L‐type calcium channel blocker verapamil markedly decreased S1P‐induced HASM cell contraction, supporting a role for calcium influx from extracellular sources. Collectively, our results suggest that S1P may regulate HASM contractility, important in the pathobiology of asthma.—Rosenfeldt, H. M., Amrani, Y., Watterson, K. R., Murthy, K. S., Panettieri, R. A., Jr., Spiegel, S. Sphingosine‐1‐phosphate stimulates contraction of human airway smooth uscle cells. FASEB J. 17, 1789–1799 (2003)