Rhodopsin's oligosaccharide chains contain predominantly two types of sugar residues: mannose and N-acetylglucosamine. In the present work, bovine and rat rhodopsin were analysed biochemically for the presence of a third sugar, galactose. Treatment of bovine rod outer segments (ROS) with galactose oxidase followed by reduction with tritium-labeled sodium borohydride revealed the presence of existing molecules of galactose on rhodopsin. Rats injected intravitreally with [3H]galactose and [14C]leucine and maintained in darkness were killed 1 hr, 6 hr, 1, 3 or 5 days following the injection. Retinas were collected for subcellular fractionation and rhodopsin from each of the fractions was purified by ConA sepharose chromatography and SDS-PAGE. During the first 6 hr, galactose selectively labeled rhodopsin in the Golgi-enriched fraction resulting in increased [3H]/[14C] ratios in both Golgi and ROS. The data suggested that trimming was occurring at the transition from Golgi to ROS. Furthermore, a decrease in isotope ratio in the ROS between 6 hr and 1 day suggested further trimming of rhodopsin after membrane assembly in the ROS. Additional in vivo experiments demonstrated existing molecules of galactose on rhodopsin's oligosaccharide chain using lectin affinity chromatography. Rats injected intravitreally with [35S]methionine were dark-adapted for 2 hr. Following subcellular fractionation of retinas, ConA purified rhodopsin from ROS was applied to one of two additional lectin columns: Ricinus communis agglutinin (RCA) or Griffonia simplicifolia I (GSA). Eight to nine percent of the labeled rhodopsin was bound to and eluted from RCA, whereas none bound to GSA, indicating the presence of a beta-galactoside. The RCA agarose eluted protein co-electrophoresed with a rhodopsin standard and was light sensitive. Galactose was shown to be the terminal sugar on this subset of rhodopsin and was not capped by neuraminic acid. Binding of rhodopsin's oligosaccharide to RCA was abolished by pre-treatment with beta-galactosidase. Decreased binding of rhodopsin to RCA was observed following intravitreal injection of castanospermine but not swainsonine. Of those two inhibitors of glycoprotein trimming, only castanospermine would be expected to prevent the addition of galactose to the oligosaccharide. The association of galactose with rat rhodopsin appeared to be a transient one. At 2 hr, 8-9% of rhodopsin contained galactose, at 6 hr only 2.2% had galactose and by 24 hr less than 1% did. The galactose was trimmed from rhodopsin's oligosaccharide presumably after its role was complete. Separation of rhodopsin of the plasma membranes from rhodopsin of discs indicated that 75% of the galactose-containing rhodopsin was in the plasma membrane and only 25% was in the discs. These findings suggested a possible role for galactose in new disc formation with subsequent removal after the discs are sealed.