Taking advantage of the improved spectral resolution of high-field electron paramagnetic resonance (EPR) at 95 GHz/3.4 T as compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the polarity profile in a protein-protein interface is obtained. Nitroxide spin label side chains are introduced at positions 88 to 94 in the AS-1 sequence of the membrane adjacent HAMP domain of the transducer protein, NpHtrII, which is reconstituted in complex with sensory rhodopsin, NpSRII fromNatronobacterium pharaonis. Position-dependent variations of the values of the nitroxide magnetic tensor componentsg_xx andA_zz suggest that the spin label side chains at positions 88 to 93 of AS-1 are located between a hydrophobic and a hydrophilic microenvironment. The observed periodicity of the polarity properties of the respective spin label microenvironment agrees with an α-helical secondary structure of this part of AS-1 and validates a recently published molecular model which locates residues 88 and 91 in the interface between helices F and G of NpSRII and AS-1 of NpHtrII close to the cytoplasmic lipid-water interface.