The ZOT N-terminal domain was expressed and refolded, yielding a soluble protein with defined secondary structure. Although distantly related to protein I of filamentous phages, no evidence of ATPase activity was found. It is therefore unlikely that the ZOT N-terminal domain is involved in cholera toxin phage packaging in Vibrio cholerae. The ZOT C-terminal domain caused delocalisation of occludin and ZO-1 from Caco-2 cell-cell contacts, irrespective of disulfide bridge formation in its putative binding domain. However, the C-terminal domain did not cause actin reorganisation and this may explain the absence of a concomitant reduction in the transepithelial electrical resistance across cell monolayers.