Phenol is an aromatic hydrocarbon that markedly increases the MgATPase activity of myosin and subfragment-1, but activates only slightly or inhibits activity in the presence of Ca²⁺ or K⁺EDTA. Pre-incubating the enzyme (10 min) with phenol in the absence of ATP reduces the MgATPase activation. We believe that the increment in steady-state MgATPase activity is caused an increment in k₊₄, similar to the effect of actin on the catalytic cycle (SBBq, 2005). Although the actin-activated S1 MgATPase in the presence of phenol (0.25, 0.5 and 1%) is greater than in control (0% phenol), the enhancement promoted by phenol is less with S1 alone. Using a cosedimentation assay we examined the effects of phenol on the affinity between actin and S1 in strong-binding states (rigor and actoS1.ADP). Phenol (0.25 and 0.5%) diminished the binding between actin and S1 in both states. For understanding of the structural mechanisms underlying actomyosin interaction and ATP hydrolysis in the presence of phenol, we investigated the intrinsic fluorescence of S1. Changes in fluorescence during pre-hydrolysis and post-hydrolysis states in the absence of actin, indicated that phenol changes the tertiary structure of S1. Fluorescence emission intensity in absence of nucleotide decrease in the presence of phenol (0.25; 0.5; 1%) and the center of mass shifted 3nn to higher wavelengths; a similar effect was obtained with S1.ADP. Thus, phenol alters S1 structure when the ATP binding site is in an open state (S1) and when the site is partially open (S1.ADP). To verify whether this drug changes the S1 tertiary structure in closed states we used ATP and two nucleotide analogs that mimic the S1*.ATP and S1.ADP.Pi states. Phenol diminished the intensity but did not change the S1 structure (center of mass) when the ATP binding site was in a closed state. Our results suggest that phenol causes an S1 structural change in some catalytic states (S1 and S1.ADP). That effect may account for the change in S1 binding to actin, and also for the enhancement of Pi release during the catalytic cycle. Financial support: CNPq, Pronex.