CHIP28 is a major water transporting protein in erythrocytes and plasma membranes in kidney proxi-mal tubule and thin descending limb of Henle. Chinese hamster ovary cells were stably transfected with the coding sequence of cloned rat kidney CHIP28k using expression vectors containing cytomegalovirus or Rous sarcoma virus promoters. Clonal cell populations expressed a 1.3-kilobase mRNA on Northern blot probed by CHIP28k cDNA and a 28-kDa protein on immunoblot probed by a polyclonal CHIP28 antibody. The clone with greatest expression produced -8 X 10’copies of CHIP28k protein/cell. Plasma membrane os-motic water permeability (P f), measured by stopped-flow light scattering, was 0.004cm/s in control (vec-tor-transfected) cells (10C) and 0.014cm/s in the CHIP28k-transfected cells. P f in CHIP28k-transfected cells had an activation energy of 4.9 kcal/mol and was reversibly inhibited by HgC12. CHIP28k expression did not affect the transport of protons and the small polar non-lectrolytes urea and formamide. CHIP28k im-munoreactivity and function was then determined in subcellular fractions. P f in 6-carboxyfluorescein-la-beled endocytic vesicles, measured by a stopped-flow fluorescence quenching assay, was 0.002cm/s (control cells) and 0.011cm/s (CHIP28k-transfected cells); Pt in transfected cells was inhibited by HgC12. Immuno-blotting of fractionated endoplasmic reticulum, Golgi, and plasma membranes revealed high densities of CHIP28k (-5000 monomers/pm2 in plasma membrane) with different glycosylation patterns; functional water transport activity was present only in Golgi and plasma membrane vesicles. Antibody detection of CHIP28k by confocal fluorescence microscopy and immunogold electron microscopy revealed localization to plasma membrane and intracellular vesicles. These studies es-tablish a stably transfected somatic cell line that strongly expresses functional CHIP28k water chan-nels. As in the original proximal tubule cells, the ex-pressed CHIP28k protein is a selective water channel that is functional in endocytic vesicles and the cell plasma membrane.