Ca²⁺/calmodulin-dependent protein kinase IV-deficient (CaMKIV^−/−) mice have been used to investigate the role of this enzyme in CD4⁺ T cells. We identify a functional defect in a subpopulation of CD4⁺ T cells, characterized by a cell surface marker profile usually found on memory phenotype CD4⁺ T cells. Upon T-cell receptor engagement, the mutant cells produce diminished levels of interleukin-2 (IL-2), IL-4, and gamma interferon protein and mRNA. The defect is secondary to an inability to phosphorylate CREB and to induce CREB-dependent immediate-early genes, including c-jun, fosB, fra2, and junB, which are required for cytokine gene induction. In contrast, stimulated naive CD4⁺ T cells from CaMKIV^−/− mice show normal CREB phosphorylation, induction of immediate-early genes, and cytokine production. Thus, in addition to defining an important signaling role for CaMKIV in a subpopulation of T cells, we identify differential signaling requirements for cytokine production between naive T cells and T cells that express cell surface markers characteristic of the memory phenotype.