The mercurial insensitive water channel (MIWC, AQP-4) is a water-selective transporter expressed at the basolateral plasma membrane of principal cells in kidney collecting duct, airway epithelium, and gastric parietal cells, as well as in astrocytes and skeletal muscle plasmalemma. Because these sites correspond to membranes where orthogonal arrays of particles (OAPs) have been observed by freeze-fracture electron microscopy, we tested the hypothesis that MIWC forms OAPs. Chinese hamster ovary cells were stably transfected with the coding sequence of rat MIWC under a cytomegalovirus promoter. Immunostaining of clonal cell populations showed MIWC expression at the plasma membrane. A single band at 31 kDa was detected on immunoblot. Cell fractionation by sucrose gradient centrifugation indicated strong MIWC expression in plasma membrane fractions with lesser expression in Golgi. Functional analysis by stopped-flow light scattering showed high mercurial insensitive water permeability in plasma membrane vesicles. Freeze-fracture electron microscopy showed distinct OAPs on the plasma membrane P-face of MIWC-expressing cells with morphology indistinguishable from that in basolateral membrane of kidney collecting duct; the E-face showed corresponding linear grooves (spacing, approximately 8 nm) in transfected cells and collecting duct. OAPs were not observed in control (empty vector-transfected) cells or CHIP28 (AQP1)-transfected cells in which disorganized intramembrane particle aggregates were found. These results provide direct evidence that a molecular water channel can spontaneously assemble in regular arrays.