A surface antigen on the mouse lymphoma cell line EL-4 has been identified by a rat monoclonal antibody. The antigen, YE1/7.1, is strongly expressed on at least two other T lymphoma cell lines, BW5147 and MBL-2, but not on several Thy-1-lymphoid cell lines. This antigen is not significantly expressed on normal adult thymocytes, spleen cells, bone marrow cells, or mitogen-activated T and B cells as tested by radioimmunoassay or fluorescence analysis using flow cytometry. Whether a small number (up to 5%) of very weakly positive cells in Con A-stimulated spleen cell populations represent a unique subpopulation is unknown. This antigen is, however, demonstrable on a subpopulation of fetal thymocytes as well as on mitogen-activated adult thymocytes. When adult thymocytes are separated by agglutination with peanut agglutinin (PNA) into PNA+ and PNA-, and stimulated with Con A in the presence of Con A culture supernatant, YE1/7.1 antigen is expressed on the majority of stimulated PNA+ cells but only weakly on a small proportion of PNA- cells. It therefore seems likely that the YE1/7.1 is a differentiation antigen expressed on a population of proliferating immature thymocytes. Immunoprecipitation of the antigen from the surface of iodinated EL-4 cells shows it to have an apparent m.w. of 60,000 under reducing and nonreducing conditions.