Abstract: Two members of the SNARE membrane fusion machinery, syntaxin 2 and endobrevin/VAMP-8, have been found to be important to cytokinesis in mammalian cells. Inhibition of syntaxin 2 and endobrevin/VAMP-8 function by over-expression of non-membrane anchored mutants of these proteins causes failure of cytokinesis leading to the formation of binucleated cells. Time-lapse microscopy shows that only midbody abscission is prevented by over-expression of these non-membrane anchored mutants, and that other cellular events preceeding midbody abscission, such as furrowing, are unaffected.