ZO-1 is a peripheral protein that plays a central role in the macromolecular assembly of tight junctions by interacting with integral proteins (occludin, claudins, JAMs) of the membrane of adjoining cells, with the actin cytoskeleton, and with nuclear factors. Human ZO-1 is expressed in all epithelia and some specialized endothelia as variable amounts of two related isoforms, which originate from the alternatively spliced mRNA transcriptsα+andα-and whose specific differential role is still unknown. Moreover, little is known about the timing of expression of ZO-1 isoforms at the protein and mRNA level. This study shows that during growth of freshly plated Caco-2 cells theα+/α-ratio increased as a result of simultaneous increase ofα+and decrease ofα-. Differences in the isoform ratio also correlated with differences in epithelium differentiation. This was determined by aminopeptidase-N measurements of cells grown on conventional substrates and on modified, micro/nano-patterned surfaces. A comparable shift of ZO-1 isoforms was not observed in other tumour cell lines of non-intestinal origin (A549, Calu-3). Pancreatic stem cells propagated without exogenous differentiation stimuli display a slight, stable prevalence of theα-isoform. Of the intestinal cell lines examined (Caco-2 and T84) only Caco-2 cells displayed a dramatic shift in isoform expression. This suggests that this tumour cell line retains to a higher degree a developmental program related with the dynamic of enterocytic differentiationin vivo.