Recent studies indicate that internal water molecules play a critical role in membrane protein function. Here we report evidence that the local environment of one or more internal water molecules in sensory rhodopsin II (SRII) is altered by interaction with its cognate transducer HtrII. The SRII-HtrII complex mediates blue-light repellent phototaxis in halophilic archaea, using a signaling pathway similar to that in bacterial chemotaxis. We studied the photocycle of SRII and a SRII-HtrII fusion complex from Natronobacterium pharaonis using low-temperature static and room temperature time-resolved Fourier transform infrared (FTIR) difference spectroscopy. When cooled to 80 K, and illuminated the protein is trapped in its K state. A shift of 2 cm−1 between SRII (3626 cm−1) and SRII-HtrII (3628 cm−1) is found for a negative OH stretching band assigned to an internal water molecule, most likely located near the active Schiff base. In contrast, the OH stretching band for this water in the K state appears at the same frequency (3619 cm−1) for both the free and complexed receptor. Similar shifts are observed upon hydration with H2O18 shifted to a lower frequency confirming these bands arise from the OH stretching mode of water. Data are also presented on the effects of lipid environment on structural changes of internal water molecules and the receptor-transducer interactions. This work was supported by National Institutes of Health GrantsR01GM069969 (to K.J.R.) and R37GM27750 (to J.L.S.), U.S. Department of Energy Grant DE-FG02-07ER15867, and the Robert A. Welch Foundation (to J.L.S.).