Depletion of tumor associated macrophages and inhibition of tumor angiogenesis have been invoked as the principle mechanisms underlying the antitumor activity of liposomal clodronate (LC). However, previous studies have not examined the effects of LC on systemic antitumor immunity. Here, we used mouse tumor models to elucidate the role of T and NK cells in the antitumor activity elicited by the systemic administration of LC. Strikingly, we found that the antitumor activity of LC is completely abolished in immunodeficient Rag1(-/-) mice. Moreover, both Cd4(-/-) and Cd8(-/-) mice as well as mice depleted of NK cells manifested a significant impaired ability to control tumor growth following LC administration. Treatment with LC did not result in an overall increase in T- or NK-cell numbers in tumors or lymphoid organs, nor was tumor infiltration with T or NK cells altered. However, T and NK cells isolated from the spleen of LC-treated mice exhibited significant increased tumor-specific secretion of interferon γ and interleukin 17 and greater cytolytic activity. We concluded that the antitumor effects of LC are largely dependent on the generation of systemic T-cell and NK- cell activity, most likely owing to the depletion of immune suppressive myeloid cell populations in lymphoid tissues. These findings suggest that the systemic administration of LC may constitute an effective means for non-specifically augmenting the antitumor activity of T and NK cells.