This study describes the monoclonal antibody 5E9 and the cell surface antigen it defines. The hybridoma cell line T3-5E9 was derived from fusion of P3 X 63/Ag8 myeloma cells and spleen cells from BALB/c mice immunized with HSB-2 cells, a human T cell line. Although binding to only 1 to 5% of peripheral blood (PB) and spleen mononuclear cells, 5E9 antibody bound to 40 to 80% of Con A-, PHA-, or PWM-activated PB cells. Moreover, 5E9 antibody bound to variable numbers of Sezary, acute myelogenous leukemia, and ALL PB leukemia cells. 5E9 antibody bound to all hematopoietic and nonhematopoietic cell lines tested, to 11 +/- 1% of thymocytes, and 40% of nucleated bone marrow cells. Under reducing conditions, immunoprecipitation studies using 5E9 antibody demonstrated 5E9 antigen to be an 90,000 m.w. glycoprotein. Under nonreducing conditions, antigen 5E9 is a disulfide-linked dimer of approximately 190,000 daltons. Sequential precipitation experiments using antibody 5E9, alpha OD heteroantiserum (raised against T ALL cells), and monoclonal antibody OKT9 demonstrated that the 3 antibody preparations recognized the same 90,000 m.w. glycoprotein. Thus, antibody 5E9 defines an 90,000 m.w. human cell surface antigen that is absent on the majority of PB mononuclear cells and is expressed on rapidly dividing normal and malignant human cells. This monoclonal antibody should be a useful marker of human cell activation.