Nonmuscle myosin II is an important component of the cytoskeleton, playing a major role in cell motility and chemotaxis. We have previously demonstrated that, on stimulation with epidermal growth factor (EGF), nonmuscle myosin heavy chain II-B (NMHC-IIB) undergoes a transient phosphorylation correlating with its cellular localization. We also showed that members of the PKC family are involved in this phosphorylation. Here we demonstrate that of the two conventional PKC isoforms expressed by prostate cancer cells, PKCβII and PKCγ, PKCγ directly phosphorylates NMHC-IIB. Overexpression of wild-type and kinase dead dominant negative PKCγ result in both altered NMHC-IIB phosphorylation and subcellular localization. We have also mapped the phosphorylation sites of PKCγ on NMHC-IIB. Conversion of the PKCγ phosphorylation sites to alanine residues, reduces the EGF-dependent NMHC-IIB phosphorylation. Aspartate substitution of these sites reduces NMHC-IIB localization into cytoskeleton. These results indicate that PKCγ regulates NMHC-IIB phosphorylation and cellular localization in response to EGF stimulation.