Background: WNK4 acts upstream of SPAK/OSR1 in the regulation of Na-K-2Cl cotransporters. Results: Here we show that WNK4 directly binds to NKCC1, and in the presence of Cab39 it stimulates NKCC1 activity. Conclusion: WNK4 regulates NKCC1 through SPAK/OSR1-dependent and SPAK/OSR1-independent pathways. Significance: We uncovered a novel mode of Na-K-2Cl cotransporter activation which involves a direct interaction of WNK4 with the cation-chloride cotransporter.