The Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) β has an essential function in dendritic spines via binding to and reorganization of the actin cytoskeleton during plasticity events not shared by CaMKIIα isoform. CaMKIIβ and CaMKIIα isoforms have remarkable structural differences within the variable region. Three exons (E1, E3, and E4) are present in CaMKIIβ but not in CaMKIIα gene. Four splice variants of CaMKIIβ isoforms (CaMKIIβ, β', βe and β'e) were discovered in embryonic and adult brains. Exons E1 (lacked in βe and β'e) and E4 (lacked in β' and β'e) are subject to differential alternative splicing. We hypothesized that the sequences encoded by exons E1, E3, and/or E4 are involved in CaMKIIβ-specific bundling to the F-actin cytoskeleton. We tested the colocalization and association of these CaMKIIβ variants within an F-actin-rich structure (microspike) in CaMKIIα free embryonic day 18 (E-18) rat cortical neurons. Our results showed that CaMKIIβ and CaMKIIβ' containing exon E1 displayed an association with F-actin, while CaMKIIβe and CaMKIIβ'e lacking E1 did not. Moreover, CaMKIIβ' lacking exon E4 but having E1 showed decreased actin bindingcapacity compared to WT CaMKIIβ. This suggested E1 is required for the association between CaMKIIβ and F-actin, while E4 assists CaMKIIβ to associate with F-actin better. Thus, alternative splicing of CaMKIIβ variants in developing neurons may serve as a developmental switch for actin cytoskeleton-associated isoforms and therefore correlated with dendritic arborization and synapse formation during LTP.