We here review mechanisms that can regulate the activity of myosin II, in smooth muscle and non‐muscle cells, by modulating the Ca²⁺ sensitivity of myosin regulatory light chain (RLC) phosphorylation. The major mechanism of Ca²⁺ sensitization of smooth muscle contraction and non‐muscle cell motility is through inhibition of the smooth muscle myosin phosphatase (MLCP) that dephosphorylates the RLC in smooth muscle and non‐muscle. The active, GTP‐bound form of the small GTPase RhoA activates a serine/threonine kinase, Rho‐kinase, that phosphorylates the regulatory subunit of MLCP and inhibits phosphatase activity. G‐protein‐coupled release of arachidonic acid may also contribute to inhibition of MLCP acting, at least in part, through the Rho/Rho‐kinase pathway. Protein kinase C(s) activated by phorbol esters and diacylglycerol can also inhibit MLCP by phosphorylating and thereby activating CPI‐17, an inhibitor of its catalytic subunit; this mechanism is independent of the Rho/Rho‐kinase pathway and plays only a minor, transient role in the G‐protein‐coupled mechanism of Ca²⁺ sensitization. Ca²⁺ sensitization by the Rho/Rho‐kinase pathway contributes to the tonic phase of agonist‐induced contraction in smooth muscle, and abnormally increased activation of myosin II by this mechanism is thought to play a role in diseases such as high blood pressure and cancer cell metastasis.