Amperometry in chromaffin cells expressing green fluorescent protein (GFP) fused to synaptosome-associated protein of 25 kDa (SNAP-25) have been used to test the involvement of single amino acids in exocytotic function, overcoming some of the limitations of studies based on Botulinum neurotoxin cleavage, as this occurs at defined sites of the protein. Constructs containing either the whole SNAP-25 polypeptide or several deleted forms lacking its C-terminal domain were heavily overexpressed in transfected cells. All GFP-fusions were located in both the cytoplasm and the plasma membrane. Although a construct containing complete SNAP-25 sustained normal secretion, removal of four or more amino acids of its C terminus greatly altered the overall rate and extent of exocytosis. Further mutational analysis proved that Leu203, the fourth residue from the C terminus, is critical for secretion. Kinetics of single granule fusions from cells expressing truncated forms showed slow onset and decay times when compared with control cells expressing full SNAP-25. Thus, these data provide direct evidence for the involvement of a specific residue of SNAP-25 in exocytosis and show that overexpression of GFP-SNAP contructs combined with single vesicle fusion measurements constitutes a powerful approach to dissect the structural elements playing a role in individual steps of the exocytotic cascade.