Transglutaminase from guinea pig liver inserts dansylcadaverine and putrescine into bovine rhodopsin in retinal disk membranes. The stoichiometry of labeling is one amine per rhodopsin molecule. Labeling with putrescine prevents the subsequent insertion of dansylcadaverine, suggesting that the same glutamine residue is labeled by these amines. The site of labeling was determined by taking advantage of the fact that rhodopsin in disk membranes is cleaved by a variety of proteolytic enzymes of differing specificity into two fragments, called F1 and F2 [Pober, J. S., and Stryer, L. (1975), J. Mol. Biol. 95, 477], Three lines of evidence indicate that the site labeled by transglutaminase is located in the protease-sensitive region between the F1 and F2 fragments. First, prior enzymatic proteolysis inhibits transglutaminase-catalyzed labeling. Second, prior transglutaminase-catalyzed labeling inhibits enzymatic proteolysis. Third, dansyl fluorescence is exhibited by F1 during the initial stage of proteolysis of labeled rhodopsin by subtilisin. The dansyl label is then excised from F1 with little change in the electrophoretic mobility of this fragment. This excised F1 fragment retains the oligosaccharide units which are known to be near the amino terminus of rhodopsin. Thus, the site labeled by dansylcadaverine is near the carboxy terminus of Fl. The distance between dansylcadaverine and 11-cis-retinal in labeled rhodopsin in the disk membrane was estimated by energy-transfer spectroscopy. The apparent distance of 61 Å calculated from a transfer efficiency of 31% and an R0′ of 53 Å shows that rhodopsin has an elongated shape in its native membrane environment. The nanosecond emission anisotropy kinetics of the dansyl fluorescence showed that the probe rotates very rapidly (ø1 = <1 ns) within a cone of half-angle 32°, followed by a slower rotational motion (øa = 140 ns). In Ammonyx LO detergent solution, øa was 5 ns, indicating that the region between the F1 and F2 units is much more flexible in detergent solution than in the disk membrane.