Calcyon is called a "cross-talk accessory protein" because the mechanism by which it enables the typically Gs-linked D1 dopamine receptor to stimulate intracellular calcium release depends on a priming step involving heterologous Gq-linked G-protein-coupled receptor activation. The details of how priming facilitates the D1R calcium response have yet to be precisely elucidated. The present work shows that calcyon is constitutively localized both in vesicular and plasma membrane compartments within HEK293 cells. In addition, surface biotinylation and luminescence assays revealed that priming stimulates a 2-fold increase in the levels of calcyon expressed on the cell surface and that subsequent D1R activation produces further accumulation of the protein in the plasma membrane. The effects of priming and D1R agonists were blocked by nocodazole implicating microtubules in the delivery of calcyon-containing vesicles to the cell surface. Accumulation of calcyon in the plasma membrane correlated well with increased intracellular calcium levels as thapsigargin mimicked, and 2-aminoethoxydiphenylborane abrogated, the effects of priming. KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII) also blocked the effects of priming and D1R agonists. Furthermore, expression of constitutively active forms of the kinase bypassed the requirement for priming indicating that CaMKII is a key effector in the Ca2+ and microtubule-dependent delivery of calcyon to the cell surface.