Syntaxin is one of the proteins involved in the exocytotic event through sequential binding to specific proteins, including SNAP25 and synaptobrevin. In a previous work in digitonin-permeabilized beta cells, we characterized the functional role of two segments: synA and synB of the H3 domain of syntaxin. As a continuation of these experiments in the present study we have initially outlined a zone of 17 residues as the very effective uncoupling element of the synA segment. Further functional studies have been accomplished in intact pancreatic beta cells with a specific myristoylated (myr) 13-mer peptide comprised in this active zone. These experiments showed a concentration-dependent inhibition of glucose-induced insulin release (IC50 = 4 microM) of this engineered peptide that was specific since a myristoylated random peptide with the same composition was ineffective. A second myristoylated 13-mer peptide comprised into the synB segment was shown to be even more potent promoting a selective inhibition of insulin release. These data show for the first time, that nutrient-induced secretory process can be specifically uncoupled in intact beta cells demonstrating at the same time that syntaxin plays a central role in this mechanism.