OBJECTIVE To characterize the expression patterns of the Na+-K+-Cl(-) cotransporter (NKCC) 1 and NKCC2, and the Na+-Cl(-) cotransporter (NCC) in the rat lens and to determine if they play a role in regulating lens volume and transparency. METHODS RT-PCR was performed on RNA extracted from fiber cells to identify sodium dependent cotransporters expressed in the rat lens. Western blotting and immunohistochemistry, using NKCC1, NKCC2, and NCC antibodies, were used to verify expression at the protein level and to localize transporter expression. Organ cultured rat lenses were incubated in Artificial Aqueous Humor (AAH) of varying osmolarities or isotonic AAH that contained either the NKCC specific inhibitor bumetanide, or the NCC specific inhibitor thiazide for up to 18 h. Lens transparency was monitored with dark field microscopy, while tissue morphology and antibody labeling patterns were recorded using a confocal microscope. RESULTS Molecular experiments showed that NKCC1 and NCC were expressed in the lens at both the transcript and protein levels, but NKCC2 was not. Immunohistochemistry showed that both NKCC1 and NCC were expressed in the lens cortex, but NCC expression was also found in the lens core. In the lens cortex the majority of labeling for both transporters was cytoplasmic in nature, while in the lens core, NCC labeling was associated with the membrane. Exposure of lenses to either hypotonic or hypertonic AAH had no noticeable effects on the predominantly cytoplasmic location of either transporter in the lens cortex. Incubation of lenses in isotonic AAH plus the NKCC inhibitor bumetanide for 18 h induced a cortical opacity that was initiated by a shrinkage of peripheral fiber cells and the dilation of the extracellular space between fiber cells in a deeper zone located some approximately 150 microm in from the capsule. In contrast, lenses incubated in isotonic AAH and the NCC inhibitor thiazide maintained both their transparency and their regular fiber cell morphology. CONCLUSIONS We have confirmed the expression of NKCC1 in the rat lens and report for the first time the expression of NCC in lens fiber cells. The expression patterns of the two transporters and the differential effects of their specific inhibitors on fiber cell morphology indicate that these transporters play distinct roles in the lens. NKCC1 appears to mediate ion influx in the lens cortex while NCC may play a role in the lens nucleus.