Xestoquinone isolated from an Okinawan sea sponge Xestospongia sapra inhibited both Ca2+- and K+ (EDTA)-ATPase but not Mg2+-ATPase of skeletal muscle myosin. The inhibition was abolished in the presence of dithiothreitol. However, xestoquinone unlike N-ethylmaleimide, a well-known sulfhydryl (SH)1 reagent markedly activated actomyosin ATPase. Modification of 2 moles of SH groups per myosin by xestoquinone caused a marked increase in the actomyosin ATPase activity. Kinetical analysis of stimulatory effects of xestoquinone indicates decrease in the actin concentrations which gives half of the maximum velocity (Vmax) of actomyosin ATPase reaction without affecting the Vmax, suggesting the increase in the affinity of myosin for actin. N-ethylmaleimide can modify both the SH1 and SH2 groups still after modification of 2 mole SH groups by xestoquinone. Xestoquinone modified myosin SH groups to cause a change in the fluorescence intensity of intrinsic tryptophan residues and circular dichroism. These results suggest that xestoquinone modifies the specific SH groups in myosin distinct from SH1 and SH2 resulting in activation of actomyosin ATPase. It is also suggested that xestoquinone strengthens the interaction between actin and myosin through conformational change in myosin molecule.