Calcium/calmodulin-dependent kinase IV (CaMKIV) is a multifunctional serine/threonine kinase that is positively regulated by two main events. The first is the binding of calcium/calmodulin (Ca(2+)/CaM), which relieves intramolecular autoinhibition of the enzyme and leads to basal kinase activity. The second is activation by the upstream kinase, Ca(2+)/calmodulin-dependent kinase kinase. Phosphorylation of Ca(2+)/CaM-bound CaMKIV on its activation loop threonine (residue Thr(200) in human CaMKIV) by Ca(2+)/calmodulin-dependent kinase kinase leads to increased CaMKIV kinase activity. It has also been repeatedly noted that activation of CaMKIV is accompanied by the generation of Ca(2+)/CaM-independent or autonomous activity, although the significance of this event has been unclear. Here we demonstrate the importance of autonomous activity to CaMKIV biological function. We show that phosphorylation of CaMKIV on Thr(200) leads to the generation of a fully Ca(2+)/CaM-independent enzyme. By analyzing the behavior of wild-type and mutant CaMKIV proteins in biochemical experiments and cellular transcriptional assays, we demonstrate that CaMKIV autonomous activity is necessary and sufficient for CaMKIV-mediated transcription. The ability of wild-type CaMKIV to drive cAMP response element-binding protein-mediated transcription is strictly dependent upon an initiating Ca(2+) stimulus, which leads to kinase activation and development of autonomous activity in cells. Mutant CaMKIV proteins that are incapable of developing autonomous activity within a cellular context fail to drive transcription, whereas certain CaMKIV mutants that possess constitutive autonomous activity drive transcription in the absence of a Ca(2+) stimulus and independent of Ca(2+)/CaM binding or Thr(200) phosphorylation.