Rhodopsin is a member of a superfamily of G-protein-coupled receptors that transduce signals across membranes. We used Fourier-transform infrared (FTIR) difference spectroscopy to study the interaction between rhodopsin and lipid bilayer upon receptor activation. A difference band at 1744 cm(-1) (+)/1727 cm(-1) (-) was identified in the FTIR-difference spectrum of rhodopsin mutant D83N/E122Q in which spectral difference bands arising from the carbonyl stretching frequencies of protonated carboxylic acid groups were removed by mutation. As the band was abolished by detergent delipidation, we suggested that it arose from carbonyl groups of phospholipid fatty acid esters. Rhodopsin and the D83N/E122Q mutant were reconstituted into various (13)C-labeled 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine vesicles and probed. The 1744-cm(-1) (+)/1727 cm(-1) (-) band could be unequivocally assigned to a change in the lipid ester carbonyl stretch upon receptor activation, with roughly equal contribution from both lipid esters. The band intensity scaled with the amount of rhodopsin but not with the amount of lipid, excluding the possibility that it was due to the bulk lipid phase. We also excluded the possibility that the lipid band represents a change in the number of boundary lipids or a general alteration in the boundary lipid environment upon formation of metarhodopsin II. Instead, the data suggest that the lipid band represents the change of a specific lipid-receptor interaction that is coupled to protein conformational changes.