Stimulation of T lymphocytes results in a rapid increase in intracellular calcium concentration ([Ca²⁺]_i) that parallels the activation of Ca²⁺-calmodulin–dependent protein kinase IV (CaMKIV), a nuclear enzyme that can phosphorylate and activate the cyclic adenosine monophosphate (cAMP) response element–binding protein (CREB). However, inactivation of CaMKIV occurs despite the sustained increase in [Ca²⁺]_i that is required for T cell activation. A stable and stoichiometric complex of CaMKIV with protein serine-threonine phosphatase 2A (PP2A) was identified in which PP2A dephosphorylates CaMKIV and functions as a negative regulator of CaMKIV signaling. In Jurkat T cells, inhibition of PP2A activity by small t antigen enhanced activation of CREB-mediated transcription by CaMKIV. These findings reveal an intracellular signaling mechanism whereby a protein serine-threonine kinase (CaMKIV) is regulated by a tightly associated protein serine-threonine phosphatase (PP2A).