Major histocompatibility complex (MHC) class II surface levels on thymocytes increase after CD99 ligation. The functional implication of the up-regulated MHC class II was assessed by engaging MHC class II on CD99-ligated cells. MHC class II engagement down-modulated surface levels of T cell receptor and MHC molecules, and inhibited apoptosis of CD99-ligated thymocytes and CEM tumor cells, antagonistic effects on the previously reported CD99 functions. The results were reproducible regardless of the order of ligation of MHC class II and CD99. We suggest that signaling via MHC class II on CD99-engaged cells might be involved in the thymic maturation process by damping CD99 ligation effects.