We report on the location of the cyclohexene ring of the retinylidene chromophore of bacteriorhodopsin projected onto the plane of the membrane. For this purpose, partially deuterated retinal was synthesized containing 11 deuterons at the following positions of the cyclohexene ring: one at C-2, two at C-4, three at C-16, three at C-17, and two at C-18. The partially deuterated retinal was incorporated biosynthetically during growth of the bacteria by using the mutant JW5, which is deficient in the synthesis of retinal. Undeuterated samples were prepared in the same way. Characterization by x-ray diffraction and absorption spectroscopy showed that these samples are identical to native purple membranes as judged by these criteria. A Fourier difference map was calculated from the differences in in-plane diffraction intensities between the deuterated and undeuterated dark-adapted membrane samples. Model calculations showed that the observed difference density had the amplitude expected for a label containing 11 deuterons. At 8.7 A resolution, the map shows one major peak with the center of mass of the deuterated ring in the interior of the molecule between helices 3, 4, 5, and 6. Based on this result and on our previous work on the location of the middle of the polyene chain, we conclude that the COOH-terminal helix G, to which retinal is attached at lysine-216, is either helix 2 or helix 6.