Naturally occurring CD4⁺CD25⁺T regulatory cells originate from the thymus and play a central role regarding the maintenance of peripheral tolerance by suppression of autoreactive T-cell populations. However, T regulatory cells can have beneficial as well as harmful effects. On the one hand, they prevent a variety of autoimmune and inflammatory diseases; but on the other hand, they concomitantly inhibit antitumor immune reactions by suppressing tumor-specific T-cell responses. Therefore, these ambivalent properties of T regulatory cells require detailed investigation especially with respect to a potential therapeutic exploitation of these cells. A prerequisite for such analyses is the isolation of pure T regulatory cells and the establishment of functional tests for the analysis of their suppressive properties, since no specific markers for CD4⁺CD25⁺T regulatory cells are known so far. In this chapter, techniques applying immunomagnetic beads have been used to establish an efficient method for isolation of human as well as murine regulatory T-cells. A combination of positive and negative selection steps using immunomagnetic beads of different sizes yields preparations of functional, active CD4⁺CD25⁺regulatory T-cells with high purity. Additionally, co-culture assays for functional characterization of isolated CD4⁺CD25⁺regulatory T-cells are described that are able to detect their suppressive properties for conventional CD4⁺T-cells with high sensitivity.