Brain ischemia is accompanied by lowering of intra- and extracellular pH down to 6.5 in the case of hyperglycemia, even down to pH 5.3. It has been shown that stroke often leads to irreversible damage of synaptic transmission. The mechanism of this phenomenon is still not clear. We investigated an influence of lowering of intra- and extracellular pH on free radical formation in synaptosomes, isolated neuronal presynaptic endings. We have used three model of acidosis. 1) Strong extracellular acidification down to pHo 6.0 corresponding to pHi decrease from 7.15 down to 6.04. 2) Moderate extracellular acidification down to pHo 7.0 corresponding to the lowering of pHi down to 6.92. 3) Intracellular acidification induced by addition of 1 mM amiloride corresponding to pHi decrease down to 6.65. We have shown that both types of extracellular acidification, but not intracellular acidification, increase DCFDA fluorescence that reflects reactive oxygen species formation. These three treatments induce the rise of the dihydroethidium fluorescence that reports synthesis of superoxide anion in synaptosomes. However, the impact of intracellular acidification on superoxide anion synthesis was less than that induced by moderate extracellular acidification, although 1 mM amiloride decreases the internal pH more strongly than lowering of extracellular pHo down to 7.0. Mitochondrial uncoupler CCCP (10 µM) and NADPH oxidase inhibitor DPI (30 µM) did not inhibit an increase of fluorescence of both dyes at pHo 6.0. In contrast, superoxide anion synthesis at pHo 7.0 was almost completely eliminated by CCCP, hence not affecting an increase of DCFDA fluorescence. Furthermore, using fluorescent dyes JC-1 and rhodamine-123, we confirmed that extracellular pH lowering, including pHo 7.0, but not intracellular acidification led to depolarization of intrasynaptosomal mitochondria. Assumingly, for moderate extracellular acidification the following chain of events is thought to be probable: proton binding to non-identified receptors, localized on the plasma membrane of neuronal terminals triggers transduction of the signal to the mitochondria where the superoxide anion radical is synthesized and then initiates the development of the oxidative stress. Action of strong extracellular acidification seems to be mediated by an inhibition of antioxidant enzymes and release of iron from proteins. We have shown that pHo lowering led to oxidative stress in neuronal presynaptic endings that might underlie the long term irreversible changing in synaptic transmission.