To cope with life-threatening high osmolarity, yeast activates the high-osmolarity glycerol (HOG) signaling pathway, whose core element is the Hog1 MAP kinase cascade. Activated Hog1 regulates the cell cycle, protein translation, and gene expression. Upstream of the HOG pathway are functionally redundant SLN1 and SHO1 signaling branches. However, neither the osmosensor nor the signal generator of the SHO1 branch has been clearly defined. Here, we show that the mucin-like transmembrane proteins Hkr1 and Msb2 are the potential osmosensors for the SHO1 branch. Hyperactive forms of Hkr1 and Msb2 can activate the HOG pathway only in the presence of Sho1, whereas a hyperactive Sho1 mutant activates the HOG pathway in the absence of both Hkr1 and Msb2, indicating that Hkr1 and Msb2 are the most upstream elements known so far in the SHO1 branch. Hkr1 and Msb2 individually form a complex with Sho1, and, upon high external osmolarity stress, appear to induce Sho1 to generate an intracellular signal. Furthermore, Msb2, but not Hkr1, can also generate an intracellular signal in a Sho1-independent manner.