Addition of H₂O₂ (0.5 mM) to Ehrlich ascites tumor cells under isotonic conditions results within 25 min in a substantial (22 ± 1 %) reduction in cell volume. The cell shrinkage is paralleled by net loss of K⁺, which was significant within 8 min, whereas no concomitant increase in the K⁺ or Cl^- conductances could be observed. The H₂O₂-induced cell shrinkage was unaffected by the presence of clofilium and clotrimazole, that block volume-sensitive and Ca²⁺-activated K⁺ channels, respectively, and unaffected by a raise in extracellular K⁺ concentration to a value which eliminates the electrochemical driving force for K⁺. On the other hand, the H₂O₂-induced cell shrinkage was impaired in the presence of the KCl cotransport inhibitor DIOA, following substitution of NO₃^- for Cl^-, and when the driving force for KCl cotransport was omitted. It is suggested that H2O2 activates electro neutral KCl cotransport in Ehrlich ascites tumor cells and not K⁺ and Cl^- channels. Addition of H₂O₂ to hypotonically exposed cells accelerates the regulatory volume decrease and the concomitant net loss of K⁺, whereas no additional increase in the K⁺ and Cl^- conductance was observed. The effect of H₂O₂ on cell volume was blocked by the serine/threonine phosphatase inhibitor calyculin A, indicating an important role of serine/threonine phosphorylation in the H₂O₂ mediated activation of KCl cotransport in Ehrlich cells. In contrast, addition of H₂O₂ to adherent cells, e.g., Ehrlich Lettre ascites cells, a subtype of the Ehrlich ascites tumor cells, and NIH3T3 mouse fibroblasts increased the K⁺ and Cl^- conductances after hypotonic cell swelling. Hence, H₂O₂ induces KCl cotransport or K⁺ and Cl^- channels in non-adherent and adherent cells, respectively.