N-Benzyl-p-toluenesulfonamide (BTS) is a small organic molecule that specifically inhibits the contraction of fast skeletal muscle fibers. To determine the mechanism of inhibition by BTS, we performed a kinetic analysis of its effects on the elementary steps of the actomyosin subfragment-1 ATPase cycle. BTS decreases the steady-state acto-S1 ATPase rate approximately 10-fold and increases the actin concentration for half-maximal activation. BTS primarily affects three of the elementary steps of the reaction pathway. It decreases the rate of P_i release >20-fold in the absence of actin and >100-fold in the presence of actin. It decreases the rate of S1·ADP dissociation from 3.9 to 0.8 s^-1 while decreasing the S1·ADP dissociation constant from 2.3 to 0.8 μM. BTS weakens the apparent affinity of S1·ADP for actin, increasing the K_d from 7.0 to 29.5 μM. ATP binding to S1, hydrolysis, and the affinity of nucleotide-free S1 for actin are unaffected by BTS. Kinetic modeling indicates that the binding of BTS to myosin depends on actin association/dissociation and on nucleotide state. Our results suggest that the reduction of the acto-S1 ATPase rate is due to the inhibition of P_i release, and the suppression of tension is due to inhibition of P_i release in conjunction with the decreased apparent affinity of S1·ADP·P_i and S1·ADP for actin.