Articular cartilage is avascular and consequently hypoxic: O₂ tensions of between 4% and 10% have been reported for the tissue (Grimshaw & Mason, 2000) and values for the deepest zones of cartilage are likely to be even lower. Routine protocols for isolating cartilage tissue necessarily involve some exposure to normoxia and, for this reason, articular cartilage is frequently studied in vitro at ambient O₂ levels (21%). However, this may be inappropriate since we have recently demonstrated that Na⁺/K⁺ pump activity is greater under hypoxic conditions (Fairfax et al., in press). In this report, we extend our study to the effects of more sustained, and therefore more physiological, restorations in O₂ tension to hypoxic levels on chondrocyte Na⁺ and H⁺ homeostasis. Cartilage slices were taken from bovine and equine metacarpophalangeal joints of animals humanely killed (under Home Office guidelines) for other purposes. Chondrocytes, isolated overnight by collagenase digestion at ambient levels of O₂, were incubated for 2 h at 21% or nominally zero O₂ before assaying ion transport at 37°C. pH_i was determined fluorimetrically using BCECF (Wilkins & Hall, 1992). Ouabain-sensitive ⁸⁶Rb influx was used as a measure of Na⁺/K⁺ pump activity, and amiloride-sensitive ²²Na⁺ influx for NHE activity. Statistical significance was determined using Student’s unpaired t test. Steady state pH_i was 6.95 ± 0.06 and 6.92 ± 0.02 under normoxic and anoxic conditions respectively (mean ± S.E.M., not significant (n.s.), n = 3 for all experiments). Na⁺/K⁺ pump activity was 41.6 ± 7.6 and 43.1 ± 2.4 nmol (10⁶ cells) h^-1 in oxygenated and deoxygenated cells (n.s.). By contrast, amiloride (100 μM)-sensitive Na⁺ influx during recovery from acid load was 72.4 ± 3.9 nmol (10⁶ cells) h^-1 in normoxia and 42.6 ± 10.0 in anoxia (p < 0.05). Na⁺ dependent acid equivalent efflux, measured fluorimetrically, decreased from 2.13 ± 0.12 in (normoxia) to 1.28 ± 0.28 mM min^-1 (anoxia; p < 0.05). Finally, bafilomycin (200nM)-sensitive acid efflux increased from 0.36 ± 0.40 to 0.91 ± 0.69 mM min^-1 (n.s.). The present results show that, despite the fall in glycolysis (the negative Pasteur effect; Lee & Urban, 1997), in longer term hypoxia Na⁺/K⁺ pump activity is protected. By contrast, NHE activity was halved. These findings have implications for ion homeostasis in articular chondrocytes in vivo and for the use of chondrocytes isolated at ambient O₂ tensions.