Lactacidosis is a common feature of ischaemic brain tissue, but its role in ischaemic neuropathology is still not fully understood. Na+/H+ exchange, a mechanism involved in the regulation of intracellular pH (pHi), is activated by low pHi. The role of Na+/H+ exchange subtype 1 was investigated during extracellular acidification and subsequent pH recovery in the absence and presence of (4‐isopropyl‐3‐methylsulphonyl‐benzoyl)‐guanidine methanesulfonate (HOE642, Cariporid), a new selective and powerful inhibitor of the Na+/H+ exchanger subtype 1 (NHE‐1). It was compared for normoxia and hypoxia in two glioma cell lines (C6 and F98). pHi was monitored by fluorescence spectroscopy using the intracellularly␣trapped␣pH‐sensitive dye 2′,7′‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF). Alterations in glial cell metabolism were characterized using high‐resolution 1H, 13C and 31P NMR spectroscopy of perchloric acid extracts. NHE‐1 contributed to glial pH regulation, especially at pathologically low pHi values. NHE‐1 inhibition with HOE642 during acidification caused exacerbated metabolic disorders which were prolonged during extracellular pH recovery. However, NHE‐1 inhibition during hypoxia protected the energy state of glial cells.