The response to hyperosmotic stress is mediated by the HOG pathway. The MAP kinase Hog1 activates several transcription factors, regulates chromatin-modifying enzymes and, through its interaction with RNA polymerase II, it directs this enzyme to osmotic stress-controlled genes. For such targeting, this kinase requires the interaction with transcription factors Hot1 and Sko1. However, phosphorylation of these proteins by Hog1 is not required for their functionality. In this study, we aim to identify the Hot1 elements involved in Hog1-binding and in the activation of transcription. Two-hybrid experiments demonstrated that the Hot1 sequence between amino acids 340 and 534 and the CD element of Hog1 are required for the interaction between the two proteins and the Hot1-dependent transcription regulation. Inside this Hot1 region, short sequence KRRRR (KR4, amino acids 381-385) is essential for the kinase binding. Our data show that another element, sequence EDDDDD (ED5, amino acids 541-546), is essential for Hot1 binding to chromatin. Under osmotic stress conditions, both Hot1 elements, Hog1-interaction KR4 and DNA-binding ED5, are involved in the appropriate recruitment of Hog1 and RNA polymerase II to genes controlled by this transcription factor. Moreover, both sequences are required for osmotolerance and KR4 is necessary for the functionality of the HOG pathway. According to several experiments described in this study, the Hot1 protein is capable of forming homodimers.