We recently showed that acidosis is protective during hypoxia and detrimental during reoxygenation. We hypothesized that the detrimental effect of acidosis during reoxygenation was due to a negative effect on mitochondrial function. Human postmitotic NT2-N neurons were exposed to 3 h of hypoxia and glucose deprivation and then reoxygenated for 0, 1, 4, 9, or 21 h. The detrimental effect of acidotic reoxygenation on metabolic activity was evident already after 1 h of reoxygenation, when MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reduction (percentage of normoxic controls) was significantly higher in cells reoxygenated with neutral compared with acidotic medium both after acidotic hypoxia (83+/-26% versus 67+/-27%, p=0.006) and after neutral hypoxia (51+/-12% versus 41+/-7%, p=0.005). Hypoxanthine, a marker of cellular energy failure, increased more with acidotic compared with neutral reoxygenation both after acidotic hypoxia (after 21 h: 7.7+/-2.7 versus 3.1+/-1.9 microM, p<0.001) and after neutral hypoxia (10.4+/-2.6 versus 7.9+/-2.8 microM, p=0.001). During hypoxia and reoxygenation, there was an earlier reduction in the activity of complex IV compared with complexes II+III, and the ratio between these complexes fell during the first hour of reoxygenation. The reduction in complex IV activity was alleviated with acidotic hypoxia. Acidosis during reoxygenation, however, had no effect on the activity of either complex IV or complexes II+III. We conclude that acidosis during hypoxia increases neuronal survival and preserves complex IV activity. Acidosis during reoxygenation has an early detrimental effect on metabolic activity, but this is not mediated through an effect on the mitochondrial complexes IV or II+III.