A large body of evidence indicates that the presence of regulatory T cells (Tregs) in tumors is associated with a dampened tumor-specific immune response and a clear negative impact on survival. Many different subsets of Tregs have been identified, which all act through similar or distinct pathways to suppress tumor-specific effector cells. The observation that tumor-infiltrating Tregs are able to recognize tumor-derived antigens and can be expanded by vaccines that primarily aim at reinforcing the effector arm of the antitumor response stresses the need to study Tregs for each type of cancer targeted by immunotherapy. Current protocols enable us to isolate and culture tumor-infiltrating Tregs. Ultimately, this will not only lead to a full comprehension of the specificity and working mechanisms of Tregs but also facilitate the development of successful interventions strategies for the immunotherapy of cancer.