Smooth muscle contraction is driven by cyclical, nucleotide-dependent changes in myosin conformation that alter its affinity for actin, produce force, and generate movement. We used a high throughput screen to identify compounds that inhibit the ATPase activity of smooth muscle myosin; optimization of the initial hit compounds has resulted in compounds with nanomolar affinity. A potent representative of this chemical series, CK-2018571, inhibits the steady-state ATPase activity of human smooth muscle myosin at low nanomolar concentrations, approximately 10-fold lower than are required to inhibit non-muscle myosin, the most closely related myosin II. Selectivity between smooth and striated myosin IIs are >100-fold. Transient kinetic studies demonstrate that CK-2018571 inhibits the myosin-catalyzed hydrolysis of the γ-phosphate group of ATP, with no effect on nucleotide binding or release from the enzyme. Actin co-sedimentation assays indicate that CK-2018571 stabilizes a weak actin-binding conformation of myosin in the presence of ATP. Consistent with this mechanism, CK-2018571 relaxes skinned rat tail artery muscle tissue at low micromolar concentrations. Importantly, this relaxation occurs regardless of whether the skinned muscle has been activated by calcium or by thiophosphorylation of the myosin regulatory light chain, supporting evidence that CK-2018571 relaxes smooth muscle tissue by direct inhibition of activated smooth muscle myosin. The ability of CK-2018571 to relax intact tracheal smooth muscle and aortic ring preparations at micromolar concentrations suggests this mechanism may prove useful in diseases of smooth muscle hyper-contractility, such as hypertension and asthma.