W3/25 antibody is the monoclonal product of a hybrid cell resulting from the fusion of a mouse myeloma cell line with spleen cells from a mouse immunized with rat thymocytes. Pure clones have been derived, and segregants free of parental myeloma chains have been isolated. Previous studies have shown that this antibody recognizes a subpopulation of T cells among rat thoracic duct lymphocytes. In the work reported here, three T-cell functions were assayed after separating rat thoracic duct lymphocytes on the fluorescence-activated cell sorter on the basis of labeling with W3/25 antibody. Two of the functional activities appeared to be completely segregated by this procedure. Thus, helper cell activity for an anti-hapten plaque-forming cell response was confined to the labeled population, whereas the allogeneic suppressive effect produced in a parental vector F1 adoptive transfer was mediated by cells in the unlabeled fraction. The third function, graft-versus-host activity, was almost entirely contained within the labeled subpopulation. It is concluded that the antigenic determinant recognized by the monoclonal antibody W3/25 is a differentiation marker for T-cell functional subpopulations.