Smooth muscle (SM) is unique in its ability to maintain force for long periods of time at low energy cost. This property is called the latch-state. One of the assumptions of the latch state model of Hai and Murphy is that myosin must first be phosphorylated in order to attach to the thin filament. However, we previously demonstrated that unphosphorylated (unPHOS) myosin can attach to unregulated actin filaments. The goal of this study was to measure the binding force of unPHOS SM myosin to tropomyosin-regulated actin filaments. A microsphere captured in a laser trap was attached to an actin filament decorated with SM tropomyosin-α and β. The filament was brought in contact with a pedestal coated with unPHOS pig antrum myosin. The pedestal was then moved away from the trap at constant velocity (0.5 μm.s-1). Despite pulling the pedestal away, the microsphere did not move until the force exerted by the trap on the microsphere was sufficient to overcome the binding force of myosin on the actin/tropomyosin filament. At this point, the microsphere sprang back to its unloaded position. The force of unbinding was calculated as the product of the trap stiffness and the maximal displacement of the trapped microsphere, as assessed by displacement of its center of mass. The average force of unbinding per myosin molecule (Funb) was obtained by dividing the measured force of unbinding by the number of myosin molecules estimated per actin filament length. We found that Funb was greater in presence (0.222 pN ± 0.018; mean±SE) than in absence (0.142 pN ± 0.019; p<0.001) of tropomyosin. These results demonstrate that tropomyosin strengthens the bond between unPHOS myosin and actin. Future studies will investigate the role of other regulatory proteins of the thin filament.