Accelerated lymphocyte homing and apoptosis have been suggested to contribute to potent immunosuppressive effects of FTY720, however, its main mechanism of action remains to be fully elucidated. Here, we examined the mode of action of FTY720 in mice. FTY720, when given at a single dose of 1 mg/kg, markedly decreased the number of peripheral blood lymphocytes (PBL) but moderately increased the lymphocyte numbers in lymph nodes (LN) and Peyer's patches (PP) in normal mice, as previously observed in rats. However, the sharp decrease in PBL numbers was also observed in aly/aly mice lacking LN and PP, indicating that this phenomenon is not explained by accelerated lymphocyte homing to LN and PP. In addition, the finding that a single administration of FTY720 did not suppress proliferative responses of T cells suggested that the PBL reduction could occur without inhibiting lymphocyte functions. However, when administered at the same dose for 2 weeks, FTY720 induced severe systemic lymphopenia, as well as marked suppression of lymphocyte proliferative responses in normal mice. The same treatment also prolonged skin allograft survival in aly/aly mice. Our results suggest that FTY720 suppresses in vivo immune functions mainly by inducing systemic lymphopenia and also by inhibiting T cell functions.