We have been searching for mechanism to induce smooth muscle contraction that is not associated with phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. We report that arachidonic acid (AraA) stimulates ATPase activity of unphosphorylated smooth muscle myosin with maximal stimulation (Rmax) of 6.84 + 0.51 relative to stimulation by the vehicle and with a harf-maximal effective concentration (EC50) of 50.3 + 4.2 μM. In presence of actin Rmax was 1.72 + 0.08 and EC50 was 26.3 + 2.3 μM. Our experiments with eicosanoids consisting of the AraA cascade suggested that they neither stimulated nor inhibited the activity. Under conditions that did not allow RLC to be phosphorylated. AraA stimulated contraction of smooth muscle tissue and culture cells with an Rmax of 1.45 + 0.07 and EC50 of 27.0 + 4.4 μM. In addition to the ATPase activities of the myosin, AraA stimulated those of heavy meromyosin, subfragument 1 (S1), S1 from which the RLC was removed, and a recombinant heavy chain consisting of the myosin head. The stimulatory effects of AraA on these preparations were about two fold. The site of AraA action was indicated to be the step-releasing iorganic phosphate (Pi) from the reaction intermediate of the myosin-ADP-Pi complex. The enhancement of Pi relese by AraA was supported by computer stimulation indicating that AraA docked in the actin- binding cleft oh the myosin motor domain. The stimulatory effect of AraA was detectable with both unphosphorylated myosin and the myosin which RLC was fully phosphorylated. The AraA effect on both myosin forms was suggested to cause excess contraction and such as vasospasm.