Autophosphorylation of CaM-kinase II produces a form of the enzyme not requiring Ca2+/calmodulin for sustained activity. We report that autophosphorylated CaM-kinase II dephosphorylates itself in the presence of ADP (termed autodephosphorylation). The dephosphorylation was unaffected by phosphatase inhibitors and was nucleotide specific, occurring with ADP but not with AMP, GTP, or GDP. P-32-ATP was produced when ADP was added to P-32-labeled CaM-kinase II, indicating that the enzyme was undergoing dephosphorylation through a reversal of the autophosphorylation reaction. ATP addition also produced loss of P-32 from the autophosphorylated enzyme while maintaining the kinase in a phosphorylated state. This indicates that the enzyme was undergoing cycles of autophosphorylation and dephosphorylation in the activated state. Autothiophosphorylated CaM-kinase II was resistant to autodephosphorylation, Site-directed mutants were used to show that Thr(286) was the predominant site dephosphorylated, Additionally, CaM-kinase II composed of beta subunits exhibited autodephosphorylation. Ca2+/CaM-independent activity expressed by the autophosphorylated alpha and beta holoenzymes was reversed following autodephosphorylation.