Maintenance of β1 integrin-mediated cell adhesion in quiescent human mammary epithelial (HME) cells requires protein phosphatase (PP) 2A for not only dephosphorylation of β1 integrin but also recruitment of IQGAP1 to Rac-bound β1 integrin. However, how PP2A-dependent regulatory machinery of cell adhesion responds to EGF remains to be elucidated. We report here that phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) at threonine 286 was involved in the β1 integrin complex that consisted of PP2A, Rac, and IQGAP1 in quiescent HME cells. Stimulation of the cells with EGF concomitantly induced an increase in intracellular Ca2+, activation of CaMKII, and dissociation of PP2A-IQGAP1-CaMKII from β1 integrin-Rac. Because the activation of CaMKII and dissociation of PP2A-IQGAP1-CaMKII were blocked by either Ca2+-chelator or CaMKII inhibitor, we therefore propose that EGF has the ability to abrogate the PP2A function in the maintenance of β1 integrin-mediated cell adhesion by dissociation of PP2A-IQGAP1-CaMKII from β1 integrin-Rac through activation of CaMKII. J. Cell. Physiol. © 2006 Wiley-Liss, Inc.