Elimination of regulatory T lymphocytes may provide a way to break self-tolerance and unleash the anti-tumor properties of circulating lymphocytes. The use of fusion proteins, which link cytotoxic molecules to receptor targets, provides one approach to this problem. This study examined the ability of a fusion protein of interleukin-2 (IL-2) and diphtheria toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to eliminate regulatory T lymphocytes based on their expression of high-affinity IL-2 receptors. Thirteen patients (12 with metastatic melanoma, 1 with metastatic renal cell carcinoma) were treated at one of the two Food and Drug Administration-approved doses of Denileukin Diftitox (seven patients at 9 μg/kg, six patients at 18 μg/kg). None of the patients experienced an objective clinical response. Foxp3 expression did not decrease significantly overall, although it did decrease minimally among patients receiving 18 μg/kg (−2.01 ± 0.618 copies of Foxp3/103 copies of β-actin; P = 0.031). Denileukin Diftitox did not decrease the suppressive ability of CD4+CD25+ cells as quantified by an in vitro co-culture suppression assay. Furthermore, the increased numbers of lymphocytes in patients resulting from treatment with IL-2 were not susceptible to Denileukin Diftitox. Administration of Denileukin Diftitox does not appear to eliminate regulatory T lymphocytes or cause regression of metastatic melanoma.