Water transport properties of the Na+-K+-2Cl- cotransporter (NKCC) were studied in cultures of pigmented epithelial cells (PE) from the ciliary body of the eye. Here, the membrane that faces upwards contains NKCCs and can be subjected to rapid changes in bathing solution composition and osmolarity. The anatomy of the cultured cell layer was investigated by light and electron microscopy. The transport rate of the cotransporter was determined from the bumetanide-sensitive component of 86Rb+ uptake, and volume changes were derived from quenching of the fluorescent dye calcein. The water permeability (Lp) of the membrane was halved by the specific inhibitor bumetanide. The bumetanide-sensitive component of the water transport exhibited apparent saturation at osmotic gradients higher than 200 mosmol l-1. Cell shrinkages produced by NaCl or KCl were smaller than those elicited by equi-osmolar applications of mannitol, indicating reflection coefficients for these salts close to zero. The activation energy of the bumetanide-sensitive component of the Lp was 21 kcal mol-1, which is four times higher than that of an aqueous pore. The data suggest that osmotic transport via the cotransporter involves conformational changes of the cotransporter and interaction with Na+, K+ and Cl-. Similar measurements were performed on immortalized cell cultures from the thick ascending limb of the loop of Henle (TALH). Given similar overall transport rates of bumetanide-sensitive 86Rb+, the NKCCs of this tissue did not contribute any bumetanide-sensitive Lp. This suggests that the cotransporters of the two tissues are either different isoforms or the same cotransporter but in two different transport modes.