At best, only trace amounts of galactose have been detected as constituents of rhodopsin as analysed by several laboratories. Nevertheless, the enzymatic galactosylation of rhodopsin proceeds readily in vitro, a process which can be catalysed by galactosyltransferases from several sources. Little information is available, however, concerning the properties of the in vitro reaction. We have examined characteristics of the latter process with the hope of shedding light on the capacity of the retina to carry out this reaction. Kinetic properties of the galactosyltransferases of bovine and embryonic chick retinas, bovine milk and rat liver-Golgi were examined using rhodopsin, opsin, N-acetylglucosamine and ovalbumin as exogenous acceptors. All of these studies demonstrated the very limited activity of the galactosyltransferases of the retina as compared to the milk and rat liver systems. The subcellular distribution of the galactosyltransferases of bovine retina was examined. The influence of compounds that might modulate the reaction was also examined. alpha-Lactalbumin, a modifier of the galactosyltransferase in milk, acted as a competitive inhibitor of the galactosylation of opsin. Analogs of vitamin A, shown to inhibit galactosyltransferases in other systems, did not have this effect on the galactosylation of opsin. Similarly, mixing experiments could not demonstrate the presence of endogenous material that inhibited the reaction in the retina. The conformation of the visual pigment was shown to influence the reaction. After bleaching by visible light, opsin was preferred over rhodopsin as an acceptor of galactose by the galactosyltransferases of bovine and embryonic chick retinas and by rat liver. This distinction was only minimally demonstrated by the milk enzyme. The galactosylation of ovalbumin was unaffected by conditions of light or dark by any of the enzymes. While the mode ratio of galactose to rhodopsin after catalysis by the milk enzyme was about 1.6, this ratio was only about 0.01 after reaction with the enzyme from bovine retina. The linkage of galactose in enzymatically galactosylated rhodopsin and opsin was beta(1-4).