The chemokine receptor CCR7 has been implicated in maintenance of thymus morphology and establishment of tolerance to self-antigens. In this study, we provide direct evidence that negative selection of maturing thymocytes is defective in CCR7-deficent mice. Impaired negative selection was observed after TCR/CD3 complex stimulation in vivo as well as in vitro and was prominent in both double-positive and semimature single positive cells (CD4(+)CD8(-)CD24(high)). It is noteworthy that thymocytes of CCR7(-/-) mice display defective negative selection in response to endogenous superantigens, demonstrating that the defect also occurs under physiological conditions. Disturbed negative selection was correlated with delayed activation kinetics and decreased calcium flux response of CCR7(-/-) thymocytes after in vitro TCR/CD3 stimulation, suggesting that an impaired response of CCR7(-/-) thymocytes via TCR-mediated signaling is responsible for defective negative selection in these mice.