The avascular nature of articular cartilage means that chondrocytes are dependent on the diffusion of O₂ from the synovial fluid and subchondral bone. As a result, cartilage has a relatively low ambient O₂ tension, estimated at between 5% and 7% O₂. In addition, synovial, and cartilage, O₂ tensions can fall to very low levels in inflammatory conditions. Low O₂ tensions can affect ion homeostasis: cells acidify and Na⁺-H⁺ exchange (NHE) is inhibited (Milner et al. 2005). Reactive oxygen species (ROS) have been implicated in the response to altered O₂ tension (Chandeel & Schumacker, 2000). In the present study, we investigated their possible role in O₂ sensitivity of NHE in articular chondrocytes. Cartilage slices were obtained postmortem from bovine and equine metacarpophalangeal joints. Chondrocytes, isolated overnight by collagenase digestion at ambient levels of O₂, were incubated for 3 h at 20% (termed normoxia) or 1% O₂ (termed hypoxia). Intracellular pH (pH_i) was then determined fluorimetrically using BCECF, using intracellular buffering to calculate proton efflux (J_H in mmol H⁺ min^-1) following cell acidification (Wilkins & Hall, 1995). Production of reactive oxygen species (ROS), estimated using dichlorofluorescein fluorescence (Chandeel & Schumacker, 2000). O₂ deprivation caused a significant acidification of steady-state pH_i and also slowed recovery of pH, through inhibition of amiloride-sensitive NHE. Under these conditions, production of ROS was reduced by 41 ± 6% (n = 7) cf values at 20% O₂ (p < 0.05). Following treatment with antimycin A (10-100 μM), ROS levels were increased from 59±9 to 84±7 (NS cf 20%). With antimycin A, J_H increased from 1.90±0.15 mmol min^-1 (p<0.05 cf 20%) to 2.51±0.17 (N.S. cf 20%), compared with 2.42±0.12 at 20% O₂. Similar findings were observed following treatment with Co²⁺ (100 μM). NHE activity could therefore be correlated with ROS levels. Finally, when exposed to the protein phosphatase inhibitor calyculin A (100 nM), NHE activity (J_H) was 3.29±0.22 at 1% O₂, compared with 3.13±0.18 at 20% O₂. Calyculin A had no effect on ROS levels. These findings suggest that NHE activity in articular chondrocytes is inhibited by low O₂ tension, with the putative signal being a reduction in ROS. The signalling cascade coupling ROS levels with NHE activity appears to act via protein phosphorylation. The importance of these results to the chondrocyte in vivo is discussed.