Two-component signaling systems play a major role in the long-term adaptation of microorganisms to changes in osmolarity, but how osmoreceptors work is not well understood. Temporal changes in solute concentration are sensed by the chemotaxis system in Escherichia coli, enabling these bacteria to avoid regions of high osmolarity. To study how osmolarity is detected in this system, we fused yellow fluorescent protein (YFP) to the C terminus of the serine or aspartate chemoreceptor, monitored the steady-state fluorescence polarization of YFP, and found that the polarization decreased substantially upon addition of osmotic agents. This decrease was due to an increase in fluorescence resonance energy transfer between YFP fluorophores in adjacent homodimers within trimers of dimers. Thus, changes in homodimer spacing and/or orientation appear to initiate osmotactic signaling.