The 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]NaBH4 reduction. 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 48 000-53 000 Mr. Unreduced samples migrated between 46 000-50 000 Mr. Surface-labeled W3/25 antigen, electrophoresed under reducing conditions, separated into two bands of 44 000 and 52 000 Mr. Metabolically labeled MRC OX-8 antigen was identified as a protein of at least two chains of 39 000 and 34 000 Mr. There was also a fainter band at approximately 67 000 Mr. Unreduced samples indicated a more complex structure with bands at 70 000, 110 000 and 165 000 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 man, and Lyt-2 in mouse.