Non-muscle myosin II molecules are actin-binding proteins with ATPase activity, this latter capacity providing the energy required for actin filament cross-linking and contraction. The activation of these molecular motors relies on direct phosphorylation at conserved sites through different protein kinases, including the Rho-associated coiled coil-containing kinase (ROCK). In the light of some recent results found in our lab, we comment on the necessity of additional regulatory mechanisms to control the subcellular distribution of non-muscle myosin II proteins to ensure their full activation.