Light chain phosphorylation is the key event that regulates smooth and non-muscle myosin II ATPase activity. Here we show that both heads of smooth muscle heavy meromyosin (HMM) bind tightly to actin in the absence of nucleotide, irrespective of the state of light chain phosphorylation. In striking contrast, only one of the two heads of unphosphorylated HMM binds to actin in the presence of ADP, and the heads have different affinities for ADP. This asymmetry suggests that phosphorylation alters the mechanical coupling between the heads of HMM. A model that incorporates strain between the two heads is proposed to explain the data, which have implications for how one head of a motor protein can gate the response of the other.A common feature of the myosin II family is that all of its members contain two heads connected to an ␣-helical coiledcoil tail. Although the single-headed subfragment of myosin (S1) 1 has always been considered a good model system for studying myosin structure and function, it is clear that some important features of whole myosin may be overlooked by studying only this model species. A prominent example is that phosphorylation-dependent regulation of actin-activated ATPase activity of smooth and vertebrate non-muscle myosin II is manifested only in subfragments that have two heads as well as a minimal length of the coiled-coil tail (1). In contrast, single-headed myosin is unregulated and always active (2). Tethering of the two heads together via the coiled-coil tail can also impose steric and mechanical constraints on the interaction of the two heads with actin. This has been explored in the case of skeletal muscle heavy meromyosin (3, 4) (HMM) but not for the regulated HMMs, where light chain phosphorylation may also be expected to affect the coupling between the two heads.Structural differences between phosphorylated and unphosphorylated HMM in the presence of ATP were recently revealed through analysis of two-dimensional crystalline arrays by image reconstruction and docking of high resolution structures into the observed density maps (5). In the phosphorylated state the two heads showed no contact with each other. In contrast, the unphosphorylated HMM showed an interaction between the two heads, with the actin-binding interface of one head bound to the converter domain of the other. This asymmetrical interaction between the unphosphorylated heads suggested a mechanism for how phosphorylation-dependent regulation of activity is achieved. One head cannot interact with actin and therefore its ATPase activity will not be enhanced, whereas the other head cannot undergo the rotation of the converter domain that is necessary to achieve phosphate release (5). This unexpected structure provided evidence that the coupling between the heads of double-headed smooth muscle heavy meromyosin is influenced by the state of light chain phosphorylation. To further understand how the two heads of smooth muscle HMM interact with each other, we undertook a kinetic analysis of HMM in both the unphosphorylated ...