Water deficit and the resulting osmotic stress affect plant growth. To understand how plant cells monitor and respond to osmotic change from water stress, we isolated a cDNA from dehydrated Arabidopsis plants. This cDNA encodes a novel hybrid-type histidine kinase, ATHK1. Restriction fragment length polymorphism mapping showed that the ATHK1 gene is on chromosome 2. The predicted ATHK1 protein has two putative transmembrane regions in the N-terminal half and has structural similarity to the yeast osmosensor synthetic lethal of N-end rule 1 (SLN1). The ATHK1 transcript was more abundant in roots than other tissues under normal growth conditions and accumulated under conditions of high or low osmolarity. Histochemical analysis of beta-glucuronidase activities driven by the ATHK1 promoter further indicates that the ATHK1 gene is transcriptionally upregulated in response to changes in external osmolarity. Overexpression of the ATHK1 cDNA suppressed the lethality of the temperature-sensitive osmosensing-defective yeast mutant sln1-ts. By contrast, ATHK1 cDNAs in which conserved His or Asp residues had been substituted failed to complement the sln1-ts mutant, indicating that ATHK1 functions as a histidine kinase. Introduction of the ATHK1 cDNA into the yeast double mutant sln1Delta sho1Delta, which lacks two osmosensors, suppressed lethality in high-salinity media and activated the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK). These results imply that ATHK1 functions as an osmosensor and transmits the stress signal to a downstream MAPK cascade.