Despite the previous description of the leukocyte differentiation antigen CD20 as B cell restricted, the findings reported here indicate that a small subset of human T cells expresses low levels of CD20 or a cross-reacting antigen. Three different CD20 monoclonal antibodies (mAb), Leu16, B1, and 1F5, reacted with the T cell subset. B cells that expressed CD20 were CD20bright and constituted an average of 9.2 ± 3.3% of adult PBL. Meanwhile, T cells that expressed CD20 were CD20dim and represented 2.4 ± 1.5% of the PBL. This population may have been overlooked in previous studies due to the low level of CD20 expression per T cell and the small size of the subset in most individuals. Blocking studies indicated that CD20 mAb binding to CD3+ cells was due to the antigen-reactive regions of the CD20 antibodies and was not a result of Fc receptor binding, or non-specific fluorochrome or protein binding. The T cell nature of the CD20dim CD3+ cells was confirmed by the rapid rise in the intracellular calcium concentration ([Ca2+]i) of CD20dim cells observed following treatment with CD3 mAb but not following treatment with anti-human immunoglobulin (Ig). Extensive three-color immunophenotypic analyses indicated that CD20dim T cells were phenotypically heterogeneous and displayed a leukocyte differentiation profile that was slightly different than that of CD20− T cells. Thus, the CD20dim T cells were more likely than CD20− T cells to be γ/δ T cell antigen receptor positive (14% vs. 3.4%), CD8+ (57% vs. 33%), and CD45RO+ (82% vs. 51%); fewer were CD38+ (5% vs. 24%) or CD4+ (35% vs. 61%). Given that most differentiation antigens expressed on leukocytes participate in the functions of the cells that express them, it is possible that CD20 plays a role on the T cells on which it is expressed, although the function(s) of the CD20 molecule on T and B cells remains unknown. © 1993 Wiley-Liss, Inc.