Leu 23 is a human cell surface differentiation Ag expressed very early during lymphoid cell activation. Resting T lymphocytes do not express Leu 23. However, expression of Leu 23 glycoprotein can be induced on the Jurkat T leukemia cell line within a few hours after stimulation with soluble anti-CD3 mAb or Con A. After removal of the stimulus Leu 23 is expressed for greater than 30 h. A total of 20 to 30% of resting human thymocytes was unexpectedly stained by anti-Leu 23 mAb. Biochemical analysis revealed that the Leu 23 Ag expressed on thymocytes is a phosphorylated disulfide-linked dimer composed of 28- and 32-kDa subunits. Within the thymus, Leu 23 is preferentially expressed on the CD3bright, mostly CD4+ CD8- or CD4- CD8+ subpopulations. By contrast, CD3dim CD4+CD8+ thymocytes expressed only very low levels of Leu 23, and CD7+ CD3- CD4- CD8- thymocytes were Leu 23-. However, Leu 23 was induced on the CD3dim thymocytes by in vitro culture of thymocytes with anti-CD3-conjugated Sepharose. The transition from CD3dim to CD3bright within the thymus may be an important differentiation stage and influence selection of the antigenic repertoire. Therefore, the ability to detect subpopulations within the thymus that express Leu 23 Ag may help to identify thymocytes activated in vivo, possibly by the functional engagement of their CD3/TCR.