Replacement of a highly conserved glycine residue on transmembrane (TM) helix 3 of bovine rhodopsin (Gly 121 ) by amino acid residues with larger side chains causes a progressive blue-shift in the max value of the pigment, a decrease in thermal stability, and an increase in reactivity with hydroxylamine. In addition, mutation of Gly 121 causes a relative reversal in the selectivity of opsin for 11-cis-retinal over all-trans-retinal. We show that the loss of function phenotypes of the G121L mutant described above can be partially reverted specifically by the mutation of Phe 261 , a residue highly conserved in all G protein-coupled receptors. For example, the double-replacement mutant G121L/F261A has spectral, chromophore-binding, and transducin-activating properties intermediate between those of G121L and rhodopsin. This rescue of the G121L defects did not occur with the other second site mutations tested. We conclude that specific portions of TM helices 3 and 6, which include Gly 121 and Phe 261 , respectively, define the chromophore-binding pocket in rhodopsin. Finally, the results are placed in the context of a molecular graphics model of the TM domain of rhodopsin, which includes the retinal-binding pocket.G protein-coupled receptors, including the visual pigment rhodopsin, have been studied extensively by site-directed mutagenesis. In many cases, the biochemical phenotype of a mutant receptor involves the loss of a particular function. Unfortunately, a loss of function can be related either directly or indirectly to a particular amino acid replacement. It is often difficult to distinguish between direct and indirect effects. As a result, such studies often do not produce significant structural or mechanistic insights. Certain strategies have evolved to circumvent the problem of interpreting loss of function phenotypes. These include the construction of chimeric receptors (1, 2), the use of biophysical methods to assay defective mutants (3, 4), and the use of genetic selection methods, when available.In the preceding paper (5), we presented a study of a highly conserved glycine residue on transmembrane (TM) 1 helix 3 of bovine rhodopsin (Gly 121 . The results show that specific portions of TM helices 3 and 6 in rhodopsin comprise specific retinal-protein contacts that define the chromophore-binding pocket. The present results are consistent with a model of rhodopsin based on data from electron microscopy studies (6), NMR and two-photon spectroscopy measurements (7-9), and a comparative analysis of G protein-coupled receptors (10). A molecular graphics model of the transmembrane domain of rhodopsin is presented that illustrates the relative proximity of the retinal chromophore to Gly 121 and Phe 261 . Since Phe 261 is highly conserved among all G protein-coupled receptors (11), these results may be relevant to understanding the molecular mechanisms of activation of G protein-coupled receptors.EXPERIMENTAL PROCEDURES TM helix 6 mutant genes were generated by substituting a 45-base pair MluI-NdeI restriction ...