Two Saccharomyces cerevisiae plasma membrane-spanning proteins, Sho1 and Sln1, function during increased osmolarity to activate a mitogen-activated protein (MAP) kinase cascade. One of these proteins, Sho1, utilizes the MAP kinase kinase kinase Ste11 to activate Pbs2. We previously used the FUS1 gene of the pheromone response pathway as a reporter to monitor cross talk in hog1 mutants. Cross talk requires the Sho1-Ste11 branch of the HOG pathway, but some residual signaling, which is STE11 dependent, still occurs in the absence of Sho1. These observations led us to propose the existence of another osmosensor upstream of Ste11. To identify such an osmosensor, we screened for mutants in which the residual signaling in a hog1 sho1 mutant was further reduced. We identified the MSB2 gene, which encodes a protein with a single membrane-spanning domain and a large presumptive extracellular domain. Assay of the FUS1-lacZ reporter (in a hog1 mutant background) showed that sho1 and msb2 mutations both reduced the expression of the reporter partially and that the hog1 sho1 msb2 mutant was severely defective in the expression of the reporter. The use of DNA microarrays to monitor gene expression revealed that Sho1 and Msb2 regulate identical gene sets in hog1 mutants. A role for MSB2 in HOG1 strains was also seen in strains defective in the two known branches that activate Pbs2: an ssk1 sho1 msb2 strain was more osmosensitive than an ssk1 sho1 MSB2 strain. These observations indicate that Msb2 is partially redundant with the Sho1 osmosensing branch for the activation of Ste11.