Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a member of the broad substrate specificity class of Ca(2+)/calmodulin (CaM)-dependent protein kinases and functions as a potent stimulator of Ca(2+)-dependent gene expression. Activation of CaMKIV is a transient, tightly regulated event requiring both Ca(2+)/CaM binding and phosphorylation of the kinase on T200 by an upstream CaMK kinase (CaMKK). Previously, CaMKIV was shown to stably associate with protein serine/threonine phosphatase 2A (PP2A), which was proposed to play a role in negatively regulating the kinase. Here we report that the Ca(2+)/CaM binding-autoinhibitory domain of CaMKIV is required for association of the kinase with PP2A and that binding of PP2A and Ca(2+)/CaM appears to be mutually exclusive. We demonstrate that inhibition of the CaMKIV/PP2A association in cells results in enhanced CaMKIV-mediated gene transcription that is independent of Ca(2+)/CaM. The enhanced transcriptional activity correlates with the elevated level of phospho-T200 that accumulates when CaMKIV is prevented from interacting with PP2A. Collectively, these data suggest a molecular basis for the sequential activation and inactivation of CaMKIV. First, in response to an increase in intracellular Ca(2+), CaMKIV binds Ca(2+)/CaM and becomes phosphorylated on T200 by CaMKK. These events result in the generation of autonomous activity required for CaMKIV-mediated transcriptional regulation. The CaMKIV-associated PP2A then dephosphorylates CaMKIV T200, thereby terminating autonomous activity and CaMKIV-mediated gene transcription.