In the course of isolating hybridomas from rats that had been immunized with anti-Ig activated murine B lymphoblasts, we have identified three new mAb reactive with the T200 or leukocyte common antigen family (CD45). One, MB4B4, reacts with T200 on all leukocytes, and immunoprecipitates all the molecules that are recognized by an existing mAb to this family. Two others, MB23G2 and MB15C11, react with a subset of T200 molecules that has a unique tissue and cellular distribution. By FACS analysis and by immunocytochemical labeling of tissue sections, MB23G2/15C11 bind to most, if not all, small B and T lymphocytes, but react weakly with macrophages and dendritic cells. Expression of the MB23G2/15C11 T200 epitope exhibits four distinct features on lymphocytes: 1) activation of CD4+ cells in the mixed leukocyte reaction results in a decreased expression of MB23G2/15C11, on a subset of 30-60% of the T-cell blasts; 2) of 16 T-helper clones examined, clones of the TH2 phenotype express moderate to high levels of MB23G2/15C11 (2.5-19-fold increase in median fluorescence over control) while the TH1 clones express low to moderate levels (1.2-6.4-fold increase in median fluorescence over control) of the antigen recognized by these mAb; 3) The MB23G2/15C11 mAb do not react with germinal center cells in tissue sections of spleen, lymph node, and Peyer's patches; 4) MB23G2/15C11 reacts primarily with a subset of large thymocytes localized to the medulla. Therefore, MB23G2/15C11 define a subset-restricted form of T200 (CD45R) on murine lymphocytes. The tissue distribution of this T200 associated epitope is distinct from previously defined CD45 antigens and suggests a role during several pathways of lymphocyte development.