A number of oxidants increase the K⁺ permeability of red blood cells (RBCs, Olivieri et al., 1993). One of these is peroxynitrite. This powerful oxidant, produced in vivo from nitric oxide and superoxide, increases both Cl^– dependent and Cl^– independent K⁺ flux (Grzelak et al., 2001). It may be involved in RBC dehydration, particularly important in sickle cell disease. Little is known, however, about how it interacts with other important regulators of K⁺ permeability. RBCs, isolated from heparinised blood samples (from consenting volunteers with ethical permission) were reacted with peroxynitrite at pH 7.15, 37^oC, c.4% haematocrit for 10 min in isotonic saline (composition in mM: NaCl 100, Na₂HPO₄ / Na₂HPO₄ 50 mM). K⁺ influx was then measured +/- Cl^– (replaced with NO₃^–) using ⁸⁶Rb⁺ was used as a K⁺ congener, and ouabain (100 μM) and bumetanide (1μM), to obviate influx via Na⁺/K⁺ pump and Na⁺-K⁺-2Cl^– cotransporter. Cl^– dependent K⁺ influx was taken as a measure of K⁺-Cl^– cotransporter (KCC) activity. K⁺ influx in control cells, or cells treated with decomposed peroxynitrite, was <0.15 mmol.(l cells.h)^-1 +/- Cl-, increasing to c.4.00 and c.3.00, respectively, with a KCC activity c.1, following 1 mM peroxynitrite. Thus, as reported, peroxynitrite increased K⁺ transport by both KCC and Cl^–-independent K⁺ pathways. After treatment with peroxynitrite, the following results were obtained: (1) KCC activity fell when glucose levels were increased, with inhibitions of 54±4% and 94±15% at 4 and 5 mM glucose, respectively (means±S.E.M., n=3 or 4); (2) when incubated in N₂ (instead of air), influxes ± Cl^– decreased by c.70% and KCC activity fell from 1.19±0.35 to zero; (3) KCC activity was zero, 1.10±0.38 and 1.66±0.26 at pH 7.4, 7.2 and 7.0, respectively; (4) KCC was unaffected by reduction in osmolality from 300 to 257 mOsm.kg^-1; and (5) pretreatment with calyculin A inhibited KCC fully, post treatment inhibited it by about 40±24%. Peroxynitrite-stimulated K⁺ transport was therefore inhibited by glucose (and also other -OH containing compounds including sucrose and MOPS, data not shown) and O₂ removal, whilst KCC activity was unaffected by cell volume but remained pH-sensitive. Functional protein phosphatase activity was required for stimulation. These findings emphasise, in particular, the interaction of peroxynitrite with protein kinase / phosphatase enzymes regulating aspects of membrane permeability, especially KCC activity.