1. We studied the regulatory mechanism of Na+ transport by hyposmolality in renal epithelial A6 cells. 2. Hyposmolality increased (1) Na+ absorption, which was detected as an amiloride-sensitive short-circuit current (INa), (2) Na+-K+ pump activity, (3) basolateral Cl- conductance (Gb,Cl), and (4) phosphorylation of tyrosine, suggesting an increase in activity of protein tyrosine kinase (PTK). 3. A Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which abolished Gb, Cl, blocked the INa by inhibiting the Na+-K+ pump without any direct effect on amiloride-sensitive Na+ channels. Diminution of Gb,Cl by Cl- replacement with a less permeable anion, gluconate, also decreased the hyposmolality-increased Na+-K+ pump activity. 4. The PTK inhibitors tyrphostin A23 and genistein induced diminution of the hyposmolality-stimulated Gb,Cl, which was associated with attenuation of the hyposmolality-increased Na+-K+ pump activity. 5. Taken together, these observations suggest that: (1) hyposmolality activates PTK; (2) the activated PTK increases Gb,Cl; and (3) the PTK-increased Gb,Cl stimulates the Na+-K+ pump. 6. This PTK-activated Gb,Cl-mediated signalling of hyposmolality is a novel pathway for stimulation of the Na+-K+ pump.