In order to study the role of calcium/calmodulin kinase II (CaMKII) in T cells, we generated transgenic mice expressing CaMKIIgammaB* (T287D), a partially calcium-independent mutant of CaMKIIgammaB. In these mice, the size of the thymus was increased 1.5- to 2-fold, at least in part due to an increase in the lifespan of double-positive (DP) thymocytes. More importantly, there was an increase in the number of T cells in the secondary lymphoid organs that had acquired an antigen-dependent memory phenotype. These T cells were bonafide memory cells as assessed by a variety of criteria. In addition, T cells from wild-type mice acquired calcium-independent CaMKII activity after several rounds of antigen-stimulated division. We propose that CaMKII controls a distinct process of activation-induced cellular differentiation.