Both Cs⁺ and NH₄⁺ alter neuronal Cl^- homeostasis, yet the mechanisms have not been clearly elucidated. We hypothesized that these two cations altered the operation of the neuronal K-Cl cotransporter (KCC2). Using exogenously expressed KCC2 protein, we first examined the interaction of cations at the transport site of KCC2 by monitoring furosemide-sensitive ⁸⁶Rb influx as a function of external [Rb⁺] at different fixed external cation concentrations (Na⁺, Li⁺, K⁺, Cs⁺, and NH₄⁺). Neither Na⁺ nor Li⁺ affected furosemide-sensitive ⁸⁶Rb influx, indicating their inability to interact at the cation translocation site of KCC2. As expected for an enzyme that accepts Rb⁺ and K⁺ as alternate substrates, K⁺ was a competitve inhibitor of Rb⁺ transport by KCC2. Like K⁺, both Cs⁺ and NH₄⁺ behaved as competitive inhibitors of Rb⁺ transport by KCC2, indicating their potential as transport substrates. Using ion chromatography to measure unidirectional Rb⁺ and Cs⁺ influxes, we determined that while KCC2 was capable of transporting Cs⁺, it did so with a lower apparent affinity and maximal velocity as compared to Rb⁺. To assess NH₄⁺ transport by KCC2, we monitored intracellular pH with a pH-sensitive fluorescent dye after an NH₄⁺-induced alkaline load. Cells expressing KCC2 protein recovered pH_i much more rapidly than untransfected cells, indicating that KCC2 can mediate net NH₄⁺ uptake. Consistent with KCC2-mediated NH₄⁺ transport, pH_i recovery in KCC2 expressing cells could be inhibited by furosemide (0.2mM) or removal of external [Cl^-]. Thermodynamic and kinetic considerations of KCC2 operating in alternate transport modes can explain altered neuronal Cl^- homeostasis in the presence of Cs⁺ and NH₄⁺.