Wild-type syndet is efficiently recruited at the plasma membrane in transfected AtT-20 cells. A deletion at the cysteine-rich domain abolishes palmitoylation, membrane binding, and plasma membrane distribution of syndet. Syndet, SNAP-25A, and SNAP-25B share four cysteine residues, of which three, Cys2, Cys4, and Cys5, are absolutely conserved in all three homologs. Mutations at any pair of cysteines within cysteines 2, 4, and 5 shift syndet from the cell surface into the cytoplasm. Thus, at least two cysteines within the conserved triplet are necessary for plasma membrane localization. Syndet C1S/C3S, with substitutions at the pair Cys1 and Cys3, distributes to the plasma membrane, a Golgi-like compartment, and the cytosol. We conclude that Cys1 and Cys3 are not absolutely necessary for membrane binding or plasma membrane localization. Our results show that the cysteine-rich domain of syndet plays a major role in its subcellular distribution.