Rhodopsin, the photoreceptor molecule of the vertebrate rod cell, is a G protein-coupled receptor. Rhodopsin consists of the opsin apoprotein and its 11-cis-retinal chromophore, which is covalently bound to a specific lysine residue by a stable protonated Schiff base linkage. Rhodopsin activation occurs when light causes photoisomerization of the 11-cis chromophore to its all-trans form. The all-trans chromophore is the receptor agonist. The 11-cis-retinylidene chromophore is analogous pharmacologically to a potent inverse agonist of the receptor. We report here that replacement of a highly conserved glycine residue (Gly121) causes 11-cis-retinal to become a pharmacologic partial agonist. Although the mutant apoproteins do not display constitutive activity, they are active in the dark when bound to an 11-cis-retinylidene chromophore, or to a "locked" chromophore analogue, Ret-7. The degree of partial agonism is directly related to the size of the amino acid replacement at position 121, and it can be reversed by a specific second-site replacement of Phe261. Thus, mutation of Gly121 in rhodopsin causes 11-cis-retinal to act as a partial agonist rather than an inverse agonist, allowing the mutant pigment to activate transducin in the dark.