Recently, we have described the presence and possible role of syntaxin in pancreatic beta-cells by using monoclonal antibodies [F. Martin, F. Moya, L. M. Gutierrez, J.A. Reig, B. Soria (1995) Diabetologia 38, 860-863]. In order to characterize further the importance of specific domains of this protein, the functional role of a particular region of the syntaxin-1 molecule has now been investigated by using two synthetic peptides, SynA and SynB, corresponding to two portions of the H3 region at the C-terminal domain of the protein, residues 229-251 and 197-219 respectively. Functional experiments carried out in permeabilized pancreatic beta-cells demonstrate that these peptides inhibit Ca(2+)-dependent insulin release in a dose-dependent manner. This effect is specific because peptides of the same composition but random sequence do not show the same effect. In contrast with this inhibitory effect on Ca(2+)-induced secretion, both peptides increase basal release. However, under the same conditions, SynA and SynB do not affect guanosine 5'-[gamma-thio]triphosphate-induced insulin release. These results demonstrate that specific portions of the H3 region of syntaxin-1 are involved in critical protein-protein interactions specifically during Ca(2+)-induced insulin secretion.