The exocytosis of neurotransmitters is regulated by calcium and is plastic - features that suggest specialized regulation of the basic membrane trafficking process. Here we show that Synaptic Vesicle Protein 2 (SV2), a protein specific to neurons and endocrine cells, is required to maintain a pool of vesicles available for calcium-stimulated exocytosis. Direct measures of exocytosis in adrenal chromaffin cells showed that the calcium-induced exocytotic burst, which operationally defines the readily releasable pool of vesicles, was significantly reduced in mice lacking SV2A. Burst kinetics were normal in cells from SV2A knockout animals, however, indicating that SV2 functions before the final events of fusion. Analyses of SDS-resistant SNARE (soluble NSF (N-ethylmaleimide-sensitive fusion) attachment protein receptor) complexes in brain tissue showed that loss of SV2A was associated with fewer SDS-resistant complexes. Our observations indicate that SV2 may modulate the formation of protein complexes required for fusion and therefore the progression of vesicles to a fusion-competent state.