This study examines the effects of 1-hexanol as a perturbing agent on actomyosin ATPase and its related functions in the concentration range between 0 and 20 mM. In this range the denaturation of myosin subfragment 1 (S1), as measured by the inactivation rate of its K-EDTA-ATPase, and depolymerization of F-actin were insignificant. Major findings showed that hexanol had the following effects which were fully reversible, (a) a marked activation of S1 MgATPase (approximately 10-fold at 20 mM) without greatly affecting the enhancement of tryptophan fluorescence by formation of S1.ADP.Pi intermediate and the rate of ADP release from S1.ADP; (b) an inhibition of the maximum actin-activated ATPase activity; (c) an increase in the affinity of S1 for actin in the presence of ATP and a decrease in the presence of ADP or the absence of nucleotide; (d) a reduction in the sliding velocity of actin filaments in in vitro motility assays with myosin, and (e) a decrease in isometric tension of single skinned muscle fibers. Thus, the effects of hexanol on actomyosin interaction are distinct for the weak and strong binding states, consistent with a change in the hydrophobic interaction in the interface between myosin and actin accompanying the transition from the weak to the strong binding state. Hexanol also accelerates the Pi release from S1.ADP.Pi, which is the transition step from the weak to the strong binding state. The fact that hexanol accelerates Pi release suggests that this alcohol perturbs the S1.ADP.Pi conformation. We speculate that this intermediate-specific structural perturbation is related to the inhibition of the maximum actin-activated ATPase, in vitro motility, and isometric tension.