The effect of oxidants on K⁺-Cl cotransport (KCC) was investigated in equine red blood cells. Carbon monoxide mimicked O₂. The substituted benzaldehyde, 12C79 (5 mM), markedly increased O₂ affinity. In N₂, however, O₂ saturation was low (<10%) but KCC remained active. Nitrite (NO₂) oxidized heme to methemoglobin (metHb). High concentrations of NO₂ (1 and 5 mM vs. 0.5 mM) increased KCC activity above control levels; it became O₂ independent but remained sensitive to other stimuli. 1-Chloro-2,4-dinitrobenzene (1-3 mM) depleted reduced glutathione (GSH). Prolonged exposure (60-120 min, 1 mM) or high concentrations (3 mM) stimulated an O₂-independent KCC activity; short exposures and low concentrations (30 min, 0.5 or 1 mM) did not. The effect of these manipulations was correlated with changes in GSH and metHb concentrations. An oxy conformation of Hb was necessary for KCC activation. An increase in its activity over the level found in oxygenated control cells required both accumulation of metHb and depletion of GSH. Findings are relevant to understanding the physiology and pathology of regulation of KCC.