iNOS expression is regulated transcriptionally in response to cytokine induction and post-translationally by palmitoylation and trafficking into perinuclear aggresomes. We investigated the effects of multifunctional calcium/calmodulin-dependent protein kinase II protein kinase (CaMKII) on iNOS trafficking in cultured rat aortic vascular smooth muscle cells (VSMC). Immunofluorescence and confocal microscopy demonstrated colocalization of iNOS and CaMKII₂ with a perinuclear distribution and concentration in aggresome-like structures identified by colocalization with -tubulin. Furthermore, CaMKII₂ coimmunoprecipitated with iNOS and pre-incubation with KN-93 (a CaMKII inhibitor) facilitated this association. Addition of Ca²⁺ mobilizing stimuli expected to activate CaMKII; a purinergic agonist (UTP) or calcium ionophore (ionomycin), caused a general redistribution of iNOS from cytosolic to membrane and nuclear fractions. Similarly, adenoviral expression of a constitutively active CaMKII₂ mutant altered iNOS localization, shifting iNOS from the cytosolic fraction. Suppression of CaMKII₂ using an adenovirus expressing a short hairpin siRNA increased nuclear iNOS localization in resting cells, but inhibited ionomycin-induced translocation of iNOS to the nucleus. Following addition of these chronic and acute CaMKII modulators there were fewer aggresome-like structures containing iNOS. All of the treatments that chronically affected CaMKII activity or expression significantly inhibited iNOS specific activity following cytokine induction. The data shows the regulation of a complex by CaMKII₂ via activation of CaMKII₂ or the loss of the kinase. This break in the complex causes a mislocalization of iNOS and abrogates iNOS activity. The results suggest that CaMKII₂ may be an important regulator of iNOS trafficking and activity in VSMCs.