Ca2 + /calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase that is best known for its role in synaptic plasticity and memory. Multiple roles of CaMKII have been identified in the hippocampus, yet its role in developing neurons is less well understood. We show here that endogenous CaMKIIβ, but not CaMKIIα, localized to prominent F-actin-rich structures at the soma in embryonic cortical neurons. Fluorescence recovery after photobleaching analyses of GFP-CaMKIIβ binding interactions with F-actin in this CaMKIIα-free system indicated CaMKIIβ binding depended upon a putative F-actin binding domain in the variable region of CaMKIIβ. Furthermore, CaMKIIα decreased CaMKIIβ binding to F-actin. We examined the interaction of CaMKIIβ with stable and dynamic actin and show that CaMKIIβ binding to F-actin was dramatically prolonged when F-actin was stabilized. CaMKIIβ binding to stable F-actin was disrupted when it was bound by Ca2 + /calmodulin or when it was highly phosphorylated, but not by kinase inactivity. Whereas CaMKIIβ over-expression increased the prevalence of the F-actin-rich structures, disruption of CaMKIIβ binding to F-actin reduced them. Taken together, these data suggest that CaMKIIβ binding to stable F-actin is important for the in vivo maintenance of polymerized F-actin.