Different radiolabeling procedures have been used in conjunction with specific immunoprecipitation to assess the mode of association of Lyt-2/3 antigens with the cell membrane. Thus, cells were labeled with two different hydrophobic probes reacting selectively with lipid-associated portions of membrane proteins. The segments of glycoproteins exposed on the outside of the plasma membrane were specifically labeled using either enzyme-catalysed surface iodination or specific labeling of the carbohydrate moiety. The results show that the three disulfide-linked polypeptides of Lyt-2/3 molecules are all surface-expressed glycopeptides possessing hydrophobic regions residing within the lipid bilayer. In particular, the 28,000 mol. wt component, barely detectable by surface iodination, can be identified as a strongly labeled homogeneous and basic species by hydrophobic, biosynthetic and glycoprotein-specific labeling procedures. In addition, differences in the expression of these components were observed between thymocytes and differentiated T-lymphocytes. Probably due to glycosylation or other processing events, the 37,000 and 32,000 mol. wt components distinguishable on thymocytes co-migrate as a broad band of apparent mol. wt 41,000-42,000 when precipitated from a cloned cytolytic T-cell line. Finally, the 28,000 mol. wt component which is abundant in thymocytes is expressed in reduced amounts on cytolytic T-cells.