To understand the domain structure responsible for different enzymatic properties, we constructed chimera cDNAs of the alpha and beta isoforms of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). The chimera DNAs were expressed in neuroblastoma cells, and the affinity for calmodulin and the subcellular localization of chimera enzymes were investigated. Here, we found that the region in immediately N-terminal of the calmodulin-binding site (exons 10 and 11), including the autophosphorylation site, mainly affected the affinity of each isoform for calmodulin and that the N-terminal region (exons 1 and 2), including the ATP-binding site, modified the affinity for calmodulin of each isoform. It was confirmed that the association of beta CaM kinase II with the particulate fraction was determined by beta-specific insertion, and also found that the association with the particulate fraction was modified by exons 1 and 2 of each isoform. Kinases without beta-specific insertion and chimera kinases consisting of exons 1 and 2 of beta and other regions of alpha appeared reduced in the transport of kinases to neurite. These results indicated that the structure of exons 10 and 11 and exons 1 and 2 modified the properties of CaM kinase II holoenzyme.