Integrins mediate cell adhesion and signaling events. The turnover and movement of adhesion sites is critical for cell migration. Ivaska et al. examined how protein kinase C ϵ (PKCϵ) influenced cell migration and B1 integrins in mouse embryonic fibroblasts deficient in PKCϵ (PKCϵKO). Cell lines in which PCKϵ had been reintroduced (PKCϵRE) exhibited more migratory behavior and more peripheral focal adhesions than did PKCϵKO cells. The increased migration toward fibronectin required the kinase activity of PCKϵ. When PKC activity was inhibited in the PKCϵRE cells by bis-indolylmaleimide I (BIM I), β1 integrin from the plasma membrane, PKCϵ, and a tetraspanin protein CD81 accumulated in intracellular vesicles, suggesting that PKCϵ regulates a step in integrin endocytosis and intracellular trafficking. Cells from the PKCϵKO did not show this same redistribution of integrins; however, after transfection of the kinase-inactive mutant of PKCϵ vesicular localization was detected. Thus, absence of PCKϵ may not be equivalent to inhibition of PKCϵ. In vitro, release of PKCϵ from vesicles isolated after BIM I treatment required energy and PKCϵ kinase activity, suggesting that PKCϵ activity regulates recycling from this compartment.