Many regulatory steps precede final membrane fusion in neuroendocrine cells. Some parts of this preparatory cascade, including fusion and priming, are dependent on the intracellular Ca(2+) concentration ([Ca(2+)](i)). However, the functional implications of [Ca(2+)](i) in the regulation of docking remain elusive and controversial due to an inability to determine the modulatory effect of [Ca(2+)](i). Using a combination of TIRF-microscopy and electrophysiology we followed the movement of large dense core vesicles (LDCVs) close to the plasma membrane, simultaneously measuring membrane capacitance and [Ca(2+)](i). We found that a free [Ca(2+)](i) of 700 nM maximized the immediately releasable pool and minimized the lateral mobility of vesicles, which is consistent with a maximal increase of the pool size of primed LDCVs. The parameters that reflect docking, i.e. axial mobility and the fraction of LDCVs residing at the plasma membrane for less than 5 seconds, were strongly decreased at a free [Ca(2+)](i) of 500 nM. These results provide the first evidence that docking and priming occur at different free intracellular Ca(2+) concentrations, with docking efficiency being the most robust at 500 nM.