The neuron-specific K⁺-Cl^-co-transporter KCC2 provides the main pathway for NH₄⁺ uptake in cultured neurons. Here we describe, that intracellular acid shifts resulting from NH₄Cl applications can be used to measure KCC2 activity in neurons. NH₄Cl application to mature cultured neurons resulted in a small initial alkalosis followed by a strong acid shift in pH_i indicating uptake of NH₄⁺. The acid shift saturated after several minutes in the presence of NH₄Cl. Removal of NH₄Cl induced a large rebound acidification. Therefore, to determine the dependence of pH_i changes on the concentration of NH₄Cl, we used brief (30s) NH₄Cl applications which allowed to compare amplitudes of rebound acid shifts. Amplitudes increased with increasing NH₄Cl concentrations and saturated above 10mM. The loop diuretic furosemide that is frequently used to inhibit KCC2 reduced NH₄Cl (7mM) induced acid shifts (half max. inhibition: 47mM furosemide). Because the loop diuretic dependent inhibition of cation-chloride cotransporters can depend on extracellular cation concentration we also tested for the concentration dependence of KCC2 inhibition by furosemide using a lower concentration of NH₄Cl (2mM). Half maximal inhibition was shifted to a higher furosemide concentration (87mM). We conclude that rebound acid shifts in response to brief NH₄Cl (<10mM) applications provide a reliable method to determine KCC2 activity in cultured neurons. Supported by the SFB 488 / D-9