The two predominant isoforms of Aquaporin-4 (AQP4), AQP4-M23 and AQP4-M1, assemble in the plasma membrane to form supramolecular structures called Orthogonal Array of Particles (OAPs) whose dimension is tightly associated to the M1/M23 ratio. Here, we explore translational regulation contribution to M1/M23 expression in primary cultures of rat astrocytes, and analyze the role of M1 mRNA 5′untranslated region (5′UTR) in this mechanism. Using isoform-specific RNAi we found that in rat astrocytes primary cultures a large proportion of M23 protein derives from M1 mRNA translation. Furthermore, site-specific mutagenesis of the 5′UTR sequence of AQP4-M1 mRNA indicates that a multiple-site leaky scanning mechanism, an out-of-frame upstream ORF (uORF), and a reinitiation mechanism are able to modulate the M1/M23 ratio and consequently, OAPs formation. These mechanisms are likely to be shared by different species, including human, and they can also be assumed to play a role in those pathophysiological situations where the organization of AQP4 in supramolecular structures (OAPs) is involved. Finally, we report that, when transfected in Hela cells, the longer rat AQP4 isoform, called Mz, which is not present in human impairs OAPs formation. © 2011 Wiley-Liss, Inc.