AbstractThe mouse monoclonal antibodies W3/25 and MRC OX‐8 have been used to distinguish rat T lymphocyte subpopulations. In the peripheral T lymphocyte population, W3/25 antibody recognizes an antigen on the Thelper(Th) subset, while MRC OX‐8 antibody recognizes an antigen on the Tsuppressor/cytotoxic(Ts/c) subset. To determine the nature of these antigens, rat thymocytes were either metabolically labeled with [35S]L‐methionine or surface‐labeled at sialic acid residues by periodate oxidation followed by [3H]NaBH4reduction. Thymocytes were solubilized with nonionic detergent, the antigens immunoprecipitated and the molecular weights determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis. Using metabolically labeled cells, W3/25 antibody immunoprecipitated an antigen that electrophoresed under reducing conditions as a broad band between 48000–53000 Mr. Unreduced samples migrated between 46000–50000 Mr. Surface‐labeled W3/25 antigen, electrophoresed under reducing conditions, separated into two bands of 44000 and 52000 Mr. Metabolically labeled MRC OX‐8 antigen was identified as a protein of at least two chains of 39000 and 34000 Mr. There was also a fainter band at approximately 67000 Mr. Unreduced samples indicated a more complex structure with bands at 70000, 110000 and 165000 Mr. Surface‐labeled MRC OX‐8 antigen was of similar nature. These data, when considered with functional and tissue distribution data, suggest that W3/25 antigen is equivalent to T4 in man and MRC OX‐8 antigen is equivalent to T8 in