Using newly generated monoclonal antibodies, we have compared the distribution of β- and γ-cytoplasmic actin in fibroblastic and epithelial cells, in which they play crucial roles during various key cellular processes. Whereas β-actin is preferentially localized in stress fibers, circular bundles and at cell-cell contacts, suggesting a role in cell attachment and contraction, γ-actin displays a more versatile organization, according to cell activities. In moving cells, γ-actin is mainly organized as a meshwork in cortical and lamellipodial structures, suggesting a role in cell motility; in stationary cells, γ-actin is also recruited into stress fibers. β-actin-depleted cells become highly spread, display broad protrusions and reduce their stress-fiber content; by contrast, γ-actin-depleted cells acquire a contractile phenotype with thick actin bundles and shrinked lamellar and lamellipodial structures. Moreover, β- and γ-actin depleted fibroblasts exhibit distinct changes in motility compared with their controls, suggesting a specific role for each isoform in cell locomotion. Our results reveal new aspects of β- and γ-actin organization that support their functional diversity.