The Escherichia coli EnvZ protein is a membrane-located osmosensor, which is a typical member of histidine kinases involved in His-Asp phosphotransfer signaling. We found that EnvZ has a leucine zipper-like motif in its presumed periplasmic domain. The functional importance of this leucine zipper-like sequence was assessed by introducing a number of appropriate amino acid substitutions. The results collectively suggest that certain leucine residues in the leucine zipper-like structure play an important role in the osmotic signal transduction mediated by EnvZ. When cysteine was substituted for the crucial leucine residues, the EnvZ dimer with disulfide bridge was detected in the cytoplasmic membrane. It was thus demonstrated that the EnvZ osmosensor exists and exerts its signaling ability as a dimer.