Occludin is an integral membrane protein located at the tight junctions of epithelial cells. Multiple domains of occludin are involved in the regulation of paracellular permeability as well as in the targeting of the protein to the tight junction. In this study, different occludin variants were identified on the mRNA level. Four differentially spliced occludin-specific mRNA transcripts were detected. Expression of the resulting proteins revealed an altered subcellular distribution and a loss of co-localization with zonula occludens protein ZO-1 in the tight junction for two of the four splice variants. Our findings demonstrate that the fourth transmembrane domain of occludin is important for targeting occludin to the tight junction. Loss of the fourth transmembrane domain leads to a relocation of the C-terminal domain to the extracellular space. The structural diversity of natural occludin variants is further increased by an additional promoter and transcription start giving rise to an alternative exon 1. Gene expression mediated by this promoter is influenced by the pro-inflammatory cytokine tumor necrosis factor alpha.