We have studied Ca2+/calmodulin-dependent protein kinase II (CaMKII) isoform distribution and activity in embryonic hippocampal neurons developing in culture. We have found a strong correlation between the expression of the alpha subunit of the enzyme and the ability to undergo depolarization-dependent phosphorylation, which in young neurons is limited to the somatodendritic pool of the kinase. The lack of responsiveness of the axons of young alphaCaMKII-positive neurons is not caused by a lower Ca2+ influx but rather by a differential balance between kinase and phosphatase activities in this compartment. After the establishment of synaptic contacts, the presynaptic pool of the kinase displays an increasing level of activity and acquires the parallel ability to phosphorylate synapsin I, which represents one of the major CaMKII presynaptic targets in mature nerve terminals. In contrast, the activity of the postsynaptic pool of the kinase remains constant throughout synaptogenesis. In the presence of a nearly homogeneous subcellular distribution, this highly regionalized regulation of activity may reflect the multifunctional roles of CaMKII in both developing and mature neurons.