Recently we obtained evidence for the existence of Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), which was partially purified in the process of the purification step of Ca2+/calmodulin-dependent protein kinase V (CaM kinase V). This enzyme promoted phosphorylation of the autophosphorylation site on CaM kinase V and activated the activity (Mochizuki, H. et al. (1993) Biochem. Biophys. Res. Commun. 197, 1595-1600). Present study revealed that CaMKK also phosphorylated CaM kinase IV associated with the activation of its activity. Phosphorylation of CaM kinase IV by CaMKK occurred on multiple sites. Furthermore, CaM kinase IV and CaM kinase V phosphorylated each other which resulted in their activation. The phosphorylation site of CaM kinase V by CaM kinase IV was the same as the autophosphorylation site. Our study suggests the existence of a heretofore CaM kinases cascade consisting of a sequential activation of each CaM kinase.