We examined the effect of urea on NaK2Cl cotransport in human erythrocytes. In erythrocytes from nine normal subjects, the addition of 45 mM urea, a concentration commonly encountered in uremic subjects, inhibited NaK2Cl cotransport by 33 +/- 7%. Urea inhibited NaK2Cl cotransport reversibly, and in a concentration-dependent fashion with half-maximal inhibition at 63 +/- 10 mM. Acute cell shrinkage increased, and acute cell swelling decreased NaK2Cl cotransport in human erythrocytes. Okadaic acid (OA), a specific inhibitor of protein phosphatase 1 and 2A, increased NaK2Cl cotransport by nearly 80%, suggesting an important role for these phosphatases in the regulation of NaK2Cl cotransport. Urea inhibited bumetanide-sensitive K influx even when protein phosphatases were inhibited with OA, suggesting that urea acted by inhibiting a kinase. In cells subjected to shrinking and OA pretreatment, maneuvers expected to increase the net phosphorylation, urea inhibited cotransport only minimally, suggesting that urea acted by causing a net dephosphorylation of the cotransport protein, or some key regulatory protein. The finding that concentrations of urea found in uremic subjects inhibited NaK2Cl cotransport, a widespread transport pathway with important physiological functions, suggests that urea is not only a marker for accumulation of other uremic toxins, but may be a significant uremic toxin itself.