AbstractSeveral monoclonal antibodies (mAb) against rabbit leukocytes were characterized in binding and functional studies. mAb 1.24 stains thymocytes, bone marrow cells, peripheral T and B cells and blood monocytes. T cells express more 1.24 antigen than B cells. In the absence of added complement (C), mAb 1.24 inhibits alloantigen‐, concanavalin A (Con A)‐, and phytohemagglutinin (PHA)‐, but not pokeweed mitogen (PWM)‐ or anti‐immunoglobulin (Ig)‐induced cell proliferation. It also strongly blocks anti‐sheep erythrocyte plaque‐forming cell responses. A second mAb, designated 4.B9, binds to 20% of thymocytes and to most, if not all, peripheral T cells andin vitro‐activated T cell blasts. A third one, 10.B3, is reactive with the nearly entire thymocyte and a major peripheral T cell population. Two‐color membrane immunofluorescence reveals the presence of a small population of peripheral blood leukocytes which bear surface Ig and are weakly stained by mAb 4.B9 and 10.B3. Without C, both 4.B9 and 10.B3 inhibit Con A‐ and PHA‐induced mitogenesis, but have no effect on PWM‐, antigen‐, or alloantigen‐induced cell proliferation. Depletion of 4.B9+cells by panning or complement lysis completely abrogates proliferative responsiveness to antigen and alloantigen, significantly reduces responsiveness to the T cell mitogens Con A and PHA, but enhances that to the B cell mitogen anti‐Ig. A fourth mAb, 12.C7, binds to 60% of thymocytes and to 10–30% of peripheral T lymphocytes at high‐level fluorescence. T cell blasts obtained in mixed leukocyte reactions are partially stained by mAb 12.C7, while those obtained after Con A or PHA activation are not. In addition, mAb 12.C7 is completely unreactive with B cells or monocytes. Without complement, it does not seem to interfere with any of thein vitrofunctions tested. All antigens studied here do not appear to be expressed in nonleukon tissues, as they do not bind to erythrocytes and are absent from brain, heart, liver and kidney as shown