Claudins provide tight junction barrier selectivity. The human CLDN5 gene contains a high-frequency single-nucleotide polymorphism (rs885985), where the G allele encodes for glutamine (Q) and the A allele encodes for an amber stop codon. Thus, these different CLDN5 alleles define nested open reading frames (ORFs) encoding claudin-5 proteins that are 303 or 218 amino acids in length. Interestingly, human claudin-16 and claudin-23 also have long ORFs. The long form of claudin-5 contrasts with the majority of claudin-5 proteins in the National Center for Biotechnology Information protein database, which are less than 220 amino acids in length. Screening of genotyped human lung tissue by immunoblot revealed only the 218–amino acid form of claudin-5 protein; the long-form claudin-5 protein was not detected. Moreover, when forcibly expressed in transfected cells, the long form of human claudin-5 was retained in intracellular compartments and did not localize to the plasma membrane, in contrast to the 218–amino acid form, which localized to intercellular junctions. This suggests that the 303–amino acid claudin-5 protein is rarely expressed, and, if so, is predicted to adversely affect cell function. Potential roles for upstream ORFs in regulating claudin-5 expression are also discussed.