Dendritic cells (DC) are professional APC, which activate the adaptive immune response. A Ca2+-calmodulin (CaM)-CaM kinase II (CaMKII) pathway regulates maturation and MHC Class II antigen presentation in human DC. The objective of this study was to characterize the mechanisms by which CaMKII modulates the levels and subcellular distribution of MHC Class II molecules. Inhibition of CaMKII via the highly specific, autoinhibitory peptide derived from the enzyme's regulatory domain resulted in rapid (60 min) and sustained (24 h) reduction of MHC Class II levels in antigen-stimulated, primary, human DC. The initial depletion of intracellular and cell surface MHC Class II was associated with its enhanced lysosomal trafficking and increased activity of specific proteases in the absence of effects on other transmembrane proteins (CD1b and CD34) or a detectable change in lysosomal degradation of exogenous protein. Inhibition of CaMKII also resulted in significant reductions in the level and stability of MHC Class II mRNA and the levels and nucleocytosolic localization of its major transcriptional regulator CIITA. These data support a model in which CaMKII regulates the levels and localization of MHC Class II protein in human DC via transcriptional, post-transcriptional, and post-translational mechanisms. These pathways are likely important to the physiologic regulation of MHC Class II as well as to its dysregulation in disease states associated with altered CaMKII function.