Detergent-solubilized plasma membranes of Con A-activated mouse spleen cells were absorbed with Sepharose-coupled rat antibodies against resting mouse lymphocytes. The unbound fraction was used to immunize a rat, and the immune spleen cells were fused with the rat myeloma Y3 . All seven rat monoclonal antibodies produced in this way strongly reacted with mitogen-activated spleen cells but only weakly or insignificantly with unstimulated spleen cells. One of the antibodies, YE3 /19.1, was studied in detail. The antibody strongly reacted with Con A- or LPS-stimulated spleen cells, but not significantly with normal adult thymocytes, spleen cells, or bone marrow cells. Unlike the transferrin receptor, which is expressed on virtually all dividing cells, the antigen defined by YE3 /19.1 was not detected on erythroblast-enriched populations or some non-T, non-B cell lines. Therefore, the antigen, termed MALA-1, seems to be specific for activated murine lymphocytes of the T and B cell lineages. Over 25% of normal adult lymph node cells were also found to express the antigen, although the antigen densities on lymph node cells were lower than those on mitogen-stimulated spleen cells. Kinetic studies of the expression of MALA-1 and the transferrin receptor on Con A-activated spleen T cells showed that both antigens are detectable within 24 hr of Con A stimulation. Although the density of the transferrin receptor on Con A blasts declined as the cell proliferation ceased, MALA-1 expression persisted. Immunoprecipitation of MALA-1 from surface-iodinated Con A blasts revealed its m.w. to be approximately 14,000 to 18,000.