Previously, we reported an mAb, GL7, that defines an activation Ag expressed by in vitro-stimulated B and T cells as well as by a subpopulation of thymocytes. The current study analyzes the GL7-expressing populations of adult and fetal thymus and demonstrates that: 1) The majority of GL7+ adult thymocytes are CD4+CD8- cells that are CD3 epsilon high, TCR-alpha beta high, HSAlow, and bimodal for CD69 expression. The 3G11-6C10- subset of CD4+CD8- thymocytes is enriched in GL7-expressing cells. 2) Strain differences exist in the expression of GL7 on adult CD4+CD8- thymocytes; 21.9 +/- 5.9% of BALB/c CD4+CD8- thymocytes are GL7+, whereas 4.4 +/- 1.7% of C57BL/6 CD4+CD8- thymocytes are GL7+. The low GL7 expression phenotype is dominant in CB6F1 thymocytes (7.0 +/- 2.0%), and analysis of BALB/c x CB6F1 mice suggests that low GL7 expression is determined by multiple genes. 3) CD4+CD8- GL7+ thymocytes from BALB/c mice, but not C57BL/6 mice, are skewed toward a high proportion of V beta 8+ cells. 4) Adult GL7+ CD4+CD8- thymocytes can be activated by TCR-specific stimuli to proliferate and to secrete high amounts of IL-4. 5) Fetal thymocytes contain GL7+ cells, which are predominantly CD4-CD8-, HSAlow, CD69-, and bimodal for TCR-gamma delta. Thus, GL7 expression defines a subpopulation of functionally competent TCR-alpha beta+ CD4+CD8- thymocytes as well as TCR-gamma delta+ and TCR- subpopulations of fetal CD4-CD8- thymocytes.