Exocytosis is a fundamental cellular process that facilitates intercellular communication, particularly in the nervous system through neurotransmitter release. This study investigates the calcium (Ca²⁺)-dependent mechanism of secretion in chromaffin cells, focusing on the relationship between intracellular Ca²⁺ concentration ([Ca²⁺]i) and exocytotic vesicle fusion. Using whole-cell patch-clamp recordings, capacitance measurements, and Ca²⁺ fluorescence imaging, we identified a biphasic Ca²⁺-dependent secretion model. The results suggest the existence of two distinct vesicular pools—small vesicles (SVs) and large vesicles (LVs)—that fuse with the plasma membrane at different [Ca²⁺]i thresholds. At low [Ca²⁺]i (≤200–300 nM), only SVs undergo exocytosis, while LVs fuse when [Ca²⁺]i surpasses this critical threshold, leading to a significantly larger exocytotic response. This vesicular model provides a mechanistic explanation for the observed nonlinear Ca²⁺-dependence of secretion, highlighting the role of differential vesicle activation in neurotransmitter release regulation. These findings offer insights into the molecular mechanisms governing synaptic plasticity and neuroendocrine signaling.