Guard cells, which form stomata in leaf epidermes, sense a multitude of environmental signals and integrate this information to regulate stomatal movements. Compared with the advanced understanding of light and water stress responses in guard cells, the molecular mechanisms that underlie stomatal CO(2) signalling have remained relatively obscure. With a high-throughput leaf thermal imaging CO(2) screen, we report the isolation of two allelic Arabidopsis mutants (high leaf temperature 1; ht1-1 and ht1-2) that are altered in their ability to control stomatal movements in response to CO(2). The strong allele, ht1-2, exhibits a markedly impaired CO(2) response but shows functional responses to blue light, fusicoccin and abscisic acid (ABA), indicating a role for HT1 in stomatal CO(2) signalling. HT1 encodes a protein kinase that is expressed mainly in guard cells. Phosphorylation assays demonstrate that the activity of the HT1 protein carrying the ht1-1 or ht1-2 mutation is greatly impaired or abolished, respectively. Furthermore, dominant-negative HT1(K113W) transgenic plants, which lack HT1 kinase activity, show a disrupted CO(2) response. These findings indicate that the HT1 kinase is important for regulation of stomatal movements and its function is more pronounced in response to CO(2) than it is to ABA or light.