To determine the role of membrane transporters in intracellular pH (pH(i)) regulation under conditions of low microenvironmental O(2), we monitored pH(i) in isolated single CA1 neurons using the fluorescent indicator carboxyseminaphthorhodafluor-1 and confocal microscopy. After total O(2) deprivation or anoxia (PO(2) approximately equal to 0 Torr), a large increase in pH(i) was seen in CA1 neurons in HEPES buffer, but a drop in pH(i), albeit small, was observed in the presence of HCO(3)(-). Ionic substitution and pharmacological experiments showed that the large anoxia-induced pH(i) increase in HEPES buffer was totally Na(+) dependent and was blocked by HOE-694, strongly suggesting the activation of the Na(+)/H(+) exchanger (NHE). Also, this pH(i) increase in HEPES buffer was significantly smaller in Na(+)/H(+) exchanger isoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons, demonstrating that NHE1 is responsible for part of the pH(i) increase following anoxia. Both chelerythrine and H-89 partly blocked, and H-7 totally eliminated, this anoxia-induced pH(i) increase in the absence of HCO. We conclude that 1) O(2) deprivation activates Na(+)/H(+) exchange by enhancing protein kinase activity and 2) membrane proteins, such as NHE, actively participate in regulating pH(i) during low-O(2) states in neurons.