Synaptotagmins are a family of membrane proteins that are characterized by a single transmembrane region and tandem C2 domains and that are likely to regulate constitutive and/or regulated vesicle traffic. We have shown that a subclass of synaptotagmins (III, V, VI, and X) forms homo- and heterodimers through an evolutionarily conserved cysteine motif at their N termini (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol Chem. 274, 31421- 31427). In this study, we identified a novel alternatively spliced variant of synaptotagmin (Syt) VI that lacks the N-terminal 85 amino acids including the transmembrane region (thus designated as Syt VIΔTM). Because it lacks the cysteine motif responsible for self-dimerization, Syt VIΔTM could not associate with Syt VI even in the presence of Ca2+. Despite lacking the transmembrane region, Syt VIΔTM can associate with the plasma membrane through the C-terminal 29 amino acids. In adult mouse brain, two closely comigrating bands at M(r) ~50,000, which closely corresponded to the molecular weight of recombinant Syt VIΔTM, were detected by anti-Syt VI antibody. These immunoreactive bands were found in both soluble and membrane fractions of mouse brain, indicating that they are membrane-associated proteins (Syt VIΔTM), but not transmembrane proteins (Syt VI). Expression of Syt VI and Syt VIΔTM in PC12 or COS-7 cells indicated that the two molecules have a distinct subcellular distribution: Syt VIΔTM is present in the cytosol or is associated with the plasma membrane or internal membrane structures, whereas Syt VI is localized to the endoplasmic reticulum and/or Golgi-like perinuclear compartment. These results suggest that Syt VI and Syt VIΔTM may play distinct roles in vesicular trafficking.