Membrane syntaxin plays essential roles in exocytosis in eukaryotic cells. The conservative H-abc domain in plasma membrane syntaxins implies important roles for syntaxin targeting and function. Our previous study showed H-abc domain was necessary for the trafficking and cluster distribution of syntaxin 1A on the plasma membrane. Here we identified which of the three domains (H-a, H-b and H-c) was essential for Stx1A trafficking and clustering. We found that, in INS-1 cells, the mutant truncated with either H-a, H-b or H-c domain could be sorted to the cell surface by a different mechanism compared to that of whole H-abc, truncated mutant. In contrast to wild type Stx1A, none of the mutants showed cluster distribution at the functional sites, suggesting that the physiological localization of Stx1A relies on intact H-abc, domain. Furthermore Munc18-1 is found not to be essential for Stx1A cluster distribution, despite important role in stabilizing membrane delivery of Stx1A. (c) 2007 Elsevier Inc. All rights reserved.