Cerebral malaria (CM) associated with Plasmodium berghei ANKA (PbA) infection is an accepted model of human CM. CM during PbA infection critically depends on sequestration of T cells into the brain. Several studies aimed to address the role of regulatory T cells (T reg) in modulating this pathogenic T cell response. However, these studies are principally hampered due to the fact that until recently no reagents were available to deplete Foxp3+ T reg specifically. To study the function of T reg in the genesis of CM, we used depletion of T reg mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin receptor-enhanced GFP fusion protein under the control of the foxp3 gene locus. These mice allow for a selective depletion of Foxp3+ T reg by diphtheria toxin injection, and also their specific detection and purification during an ongoing infection. Using depletion of T reg mice, we found only a small increase in the absolute numbers of Foxp3+ T reg during PbA infection and, consequently, the ratio of T reg to T effector cells (T eff) decreased due to the rapid expansion of T eff. Although the latter sequester in the brains of infected mice, almost no T reg were found in the brains of infected mice. Furthermore, we demonstrate that depletion of T reg has no influence on sequestration of T eff and on the clinical outcome, and only minor influence on T cell activation. Using ex vivo analysis of purified T reg from either naive mice or PbA-infected mice, we found that both exhibit similar inhibitory capacity on T eff.