The aim of this study was to elucidate the mechanism responsible for lymphopenia after exercise. Seven young healthy men volunteered for this study. Peripheral blood mononuclear cells (PBMC) were cultured with cortisol and analyzed for CXCR4 expression by flow cytometry. To determine the effects of exercise, subjects performed exhaustive cycling exercise. PBMC were cultured with plasma obtained before and after the cycling exercise. Alternatively, PBMC obtained before and after exercise was cultured without plasma or glucocorticoid to examine whether PBMC were primed in vivo for CXCR4 expression. We analyzed cortisol- or plasma-treated PBMC to determine their ability to migrate through membrane filters in response to stromal-derived factor 1 (SDF-1 )/CXCL12. Cortisol dose- and time-dependently augmented CXCR4 expression on T lymphocytes, with less than 6 h of treatment sufficient to augment CXCR4 on T lymphocytes. Post-exercise plasma also augmented CXCR4 expression. Cortisol or post-exercise plasma treatment markedly enhanced migration of T lymphocyte towards CXCL12. Augmentation of CXCR4 on T lymphocytes by cortisol or plasma was effectively blocked by the glucocorticoid receptor antagonist RU486. Thus, exercise-elicited endogenous cortisol effectively augments CXCR4 expression on T lymphocytes, which may account for lymphopenia after exercise.