Although most thymocytes express high levels of Fas antigen (CD95), the role of Fas in apoptosis signaling during thymocyte maturation has not been defined. Fas apoptosis occurs primarily in the CD4+CD8+ subpopulations of thymocytes. Fas expression and apoptosis function were investigated in the CD4-8-, CD4+8+, and CD4+ and CD8 single positive thymocyte subpopulations by in vivo injection of anti-Fas and in vitro incubation of Fas with thymic organ cultures. Fas was first expressed on CD4-8- thymocytes coincident with expression of IL-2R and CD44. In Fas mutant lpr/lpr mice, defective Fas expression correlated with overproduction of late-stage CD4-8(-)-thymocytes. Fas was highly expressed on CD3dull and CD3bright thymocytes. CD4+8+CD3dull thymocytes were sensitive to Fas apoptosis, whereas more mature CD4+8+CD3bright thymocytes were resistant to Fas apoptosis. Anti-Fas incubation with established thymic organ culture for 24 hr resulted in apoptosis of approximately 25% of thymocytes. Continued incubation of thymic organ culture with anti-Fas resulted in an apoptosis rate of 25% of CD4+CD8+ thymocytes per day for the first 3 days of culture. Continued culture for further time points up to 6 days did not result in further apoptosis of the CD4+CD8+ thymocytes. These results suggest that CD4-CD8-CD44+ IL-2R+ thymocytes express Fas and there is overpopulation of the subsequent developmental stage of thymocytes in Fas mutant lpr mice. Also, early-stage CD4+8+ thymocytes are susceptible to Fas apoptosis, whereas Fas apoptosis resistance is required after 3 days of thymic organ culture. We conclude that these two populations of thymocytes are susceptible to Fas ligand-mediated apoptosis during T cell development in the thymus.