The 240-bp α exon of the tight junction (TJ) protein ZO-1 pre-mRNA is alternatively spliced. Expression of both ZO-1α+/ZO-1α− isoforms results in hermetic TJs, and these become leaky when ZO-1α− expression prevails. The α exon inclusion/skipping mechanism was studied by in vivo RT-PCR splicing assays in neural and epithelial cells, utilizing a canine minigene construct containing the α exon, and the flanking introns and exons. Inclusion of the α exon always occurs in wild-type MDCK cells and it is detectable in transfected HeLa cells. However, the α exon is skipped in transfected neural cells. Accordingly, both 5′ and 3′ splice sites surrounding the α exon appear to be suboptimal and no cis-acting splicing control elements were found in this exon. Deletion analysis revealed an 83-bp splicing enhancer in the downstream exon and a 35-bp splicing silencer at the beginning of the upstream exon. In epithelial cells all constructs rendered α exon inclusion. We conclude that, in neural cells, skipping of the α exon depends on two antagonistic exonic elements located in the flanking constitutive exons.