Acidosis coupled to lactate accumulation, called lactacidosis, occurs in cerebral ischemia or trauma and is known to cause persistent swelling in neuronal and glial cells. It is therefore possible that mechanisms of cell volume regulation are impaired during lactacidosis. Here we tested this possibility using neuronally differentiated NG108-15 cells. These cells responded to a hypotonic challenge with osmotic swelling followed by a regulatory volume decrease (RVD) under physiological pH conditions in the absence of lactate. Under normotonic conditions, sustained cell swelling without subsequent RVD was induced by exposure to lactate-containing solution with acidic pH (6.4 or 6.2), but not with physiological pH (7.4). Under whole-cell patch-clamp, osmotic swelling was found to activate outwardly rectifying Cl(-) currents in cells exposed to control hypotonic solution. A Cl(-) channel blocker, NPPB, inhibited both RVD and the swelling-activated Cl(-) current. RVD and the volume-sensitive Cl(-) current were also markedly inhibited by lactacidosis (pH 6.4 or 6.2), but neither by application of lactate with physiological pH (7.4) nor by acidification without lactate (pH 6.2). RT-PCR analysis showed mRNA expression of two isoforms of proton-coupled monocarboxylate transporters, MCT1 and MCT8, in differentiated NG108-15 cells. Thus, we conclude that persistence of neuronal cell swelling under lactacidosis is coupled to an impairment of the activity of the volume-sensitive Cl(-) channel and to dysfunction of RVD. It is also suggested that the volume-sensitive Cl(-) channel is inhibited by intracellular acidification induced by MCT-mediated proton influx under lactacidosis.