Updated project metadata. The stress-activated protein kinase Hog1 is best known for its role in osmotic stress but is also activated by a variety of mechanistically distinct environmental stressors including heat shock, ER stress, and arsenic. In the osmotic stress response, the signal is sensed upstream and relayed to Hog1 via a kinase cascade. Here we identify a novel mode of Hog1 function whereby Hog1 senses arsenic though direct physical interaction requiring three conserved cysteine residues located adjacent to the catalytic loop. These residues are essential for Hog1's role in protecting against arsenic, but completely dispensable for osmotic stress, and mediate Hog1's cellular localization upon arsenic exposure. Hog1 in turn regulates arsenic detoxification by phosphorylating the transcription factor Yap8, promoting its nuclear localization, and stimulating transcription of its only known targets, Arr2 and Arr3, which promote arsenic efflux. Arsenic binding was also observed with the related human kinases Erk1 and Erk2, suggesting that this may be a conserved aspect of this kinase family. These data provide a mechanistic basis for understanding how stress-activated kinases can sense individual threats and carry out highly specific adaptive responses.