Chondrocytes of articular cartilage experience a relatively low ambient O₂ tension, between 5% and 7% O₂. O₂ tensions may fall further to very low levels in joint disorders such as chronic inflammatory arthritis. Hypoxia therefore presents an important stress for articular chondrocytes. Low O₂ tension (1%) results in intracellular acidification and inhibition of membrane NHE (Milner et al. 2005) and reduces the levels of reactive oxygen species (ROS). Here we investigate the site of production of ROS. Cartilage slices were obtained postmortem from equine fetlock joints. Chondrocytes, isolated overnight by collagenase digestion at ambient levels of O₂, were incubated for 3 hours at 20% (termed normoxia) or 1% O₂ (termed hypoxia). Intracellular pH was determined using BCECF and intracellular buffering to calculate proton efflux (J_H) following cell acidification (Wilkins & Hall, 1995). Production of reactive oxygen species (ROS) was estimated using dichlorofluorescein (DCF) fluorescence (Chandeel & Schumacker, 2000). Rotenone (1-100μM), an inhibitor of mitochondrial complex I, caused a reduction in ROS levels: 97±8, 59±3 and 36±3% cf control (means±SEM, n=3), at 1, 10 and 100μM. Myxothiazol (0.1-10μM), which inhibits mitochondrial complex III upstream of the ubisemiquinone, the site of production of ROS, was also tested. Results were similar to those with rotenone, e.g. at 10 μM myxothiazol decreased ROS to 45±10% (3). At both 20% and 1% O₂, both rotenone and myxothiazol also inhibited NHE activity (e.g. at 40μM, rotenone inhibited J_H at 1% O₂ from 1.59±0.09 mmol/min to 1.12±0.12, n=3). These mitochondrial inhibitors, therefore, behave differently to antimycin A, which blocks complex III downstream of ubisemiquinone, increases ROS production and also abolishes the hypoxic-induced reduction in NHE activity (see accompanying poster). Diphenyleneiodonium (DPI), which inhibits flavocytochromes, including the non-mitochondrial NADPH oxidase, as well as complex I NADH dehydrogenase, also inhibited ROS levels (at 1% O₂, ROS decreased from 77±7% to 41±2%, n=3). In addition, DPI abolished the protective effects of antimycin A on hypoxic inhibition of ROS and NHE activity when both inhibitors were added together (at 1% O₂ J_H declined further from 2.52±0.15 mmol/min with 50 μM antimycin A alone, to 1.72±0.22 mmol/min with both antimycin A and 10μM DPI, n=3). These findings implicate the mitochondria, and, in particular complex III, as the site of production of ROS in articular chondrocytes. Inhibition of ROS production in hypoxia is associated with decrease in activity of NHE.