Alteration in cell volume of vertebrates results in activation of volume-sensitive ion flux pathways. Protein phosphorylation and dephosphorylation have been reported to play a crucial role in the control of volume-sensitive ion flux pathways. Exposing Amphiuma RBCs to phorbol esters in isotonic medium results in a simultaneous, dose-dependent activation of both Na⁺/H⁺ and K⁺/H⁺ exchangers (Cala, 1986). We tested the hypothesis that in Amphiuma RBCs, both shrinkage-induced Na⁺/H⁺ exchange and swelling-induced K⁺/H⁺ exchange are activated by phosphorylation-dependent reactions. To this end, we assessed the effect of calyculin A, a phosphatase inhibitor, on the activity of the aforementioned exchangers in Amphiuma RBCs. We found that exposure of Amphiuma RBCs to calyculin-A in isotonic media results in simultaneous, 1-2 order of magnitude increase in the activity of both K⁺/H⁺ and Na+/H+ exchangers. We also demonstrate that, in isotonic media, calyculin A-dependent increases in net Na uptake and K loss are a direct result of phosphatase inhibition and are not dependent upon changes in cell volume. Whereas calyculin A exposure in the absence of volume changes results in stimulation of both the Na⁺/H⁺ and K⁺/H⁺ exchangers, superimposing cell swelling or shrinkage and calyculin A treatment results in selective activation of K⁺/H⁺ or Na⁺/H⁺ exchange, respectively. We conclude that kinase-dependent reactions are responsible for Na⁺/H⁺ and K⁺/H⁺ exchange activity while undefined volume-dependent reactions confer specificity and coordinated control.