PH is a highly variable environmental factor for the root, and plant cells can modify apoplastic pH for nutrient acquisition and in response to extracellular signals. Nevertheless, surprisingly few effects of external pH on plant gene expression have been reported. We have used microarrays to investigate whether external pH affects global gene expression. In Arabidopsis thaliana roots, 881 genes displayed at least twofold changes in transcript abundance 8 h after shifting medium pH from 6.0 to 4.5, identifying pH as a major affector of global gene expression. Several genes responded within 20 min, and gene responses were also observed in leaves of seedling cultures. The pH 4.5 treatment was not associated with abiotic stress, as evaluated from growth and transcriptional response. However, the observed patterns of global gene expression indicated redundancies and interactions between the responses to pH, auxin and pathogen elicitors. In addition, major shifts in gene expression were associated with cell wall modifications and Ca2+ signalling. Correspondingly, a marked overrepresentation of Ca2+/calmodulin-associated motifs was observed in the promoters of pH-responsive genes. This strongly suggests that plant pH recognition involves intracellular Ca2+. Overall, the results emphasize the previously underappreciated role of pH in plant responses to the environment.