Cells respond to physical and chemical stimulations mediated by pH, osmolarity, and oxidative and mechanical stresses. Various signal transduction pathways cooperate and participate in these responses. Here we describe the role of c-Jun NH2-terminal kinase (JNK) in regulation of gene transcription after an increase in extracellular H+. When cells were incubated in low pH medium, the promotion of JNK phosphorylation and c-Jun expression was clearly observed in cells in an extracellular pH- and time-dependent manner. Activation of p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) was extremely weak compared with that of JNK. An increase in extracellular H+ led to enhanced nuclear translocation of phosphorylated JNK leading to augmentation of the transcriptional activity of c-Jun. Nimodipine, a blocker of voltage-gated Ca2+ ion channels, prevented the phosphorylation of JNK and expression of c-Jun in a dose-dependent manner. These results suggest a novel intracellular signalling pathway for H+-induced c-Jun expression: an increase of extracellular H+ induces JNK phosphorylation and c-Jun expression via partly extracellular Ca2+ influx through voltage-gated Ca2+ channels.