Osmotic stress is one of the severest environmental pressures for plants, commonly occurring under natural growing condition due to drought, salinity, cold and wounding. Plants sensitively respond to these stresses by activating a set of genes, which encode proteins necessary to overcome the crises. We screened such genes from tobacco plants, and identified a particular clone, which encoded a 45 kDa protein kinase belonging to the plant receptor-like cytoplasmic protein kinase class-VII, NAK (novel Arabidopsis protein kinase) group. The clone was consequently designated as NtNAK (Nicotiana tabacum AK, accession number: DQ447159). GFP-NtNAK fusion protein was localized in both cytoplasm and nucleus, and bacterially expressed NtNAK exhibited in vitro kinase activity. Its transcripts were clearly induced upon treatments of leaves with salt, mannitol, low temperature and also with abscisic and jasmonic acids and ethylene. These properties indicated NtNAK to be a typical osmo-stress-responsive protein kinase. Its target protein(s) were then screened by the yeast two-hybrid system, and one clone encoding a 32 kDa protein was identified. The protein resembled a potato stress-responsive protein CK251806, and designated as NtCK25 (accession number: DQ448851). Bacterially expressed NtCK25 was phosphorylated by NtNAK, and NtCK25-GFP fusion protein was exclusively localized in nucleus. The structure of NtCK25 was found to be similar to a human nuclear body protein, SP110, which is involved in DNA/protein binding regulation. This suggested that, perceiving osmo-stress signal, NtNAK phosphorylates and activates NtCK25, which might function in regulation of nucleus function. The present study thus suggests that NtNAK/NtCK25 constitutes a novel phosphorylation pathway for osmotic-stress response in plants.