Interaction of ICOS with its ligand (ICOSL, B7-H2) promotes T cell responses. As CD4+ CD25 high Foxp3+ naturally occurring T regulatory cells in melanoma patients express ICOS, we investigated the impact of ICOS on naturally occurring T regulatory cell function. Expression of ICOS and T regulatory (Treg) cell markers was determined on CD4+ CD25 high T cells in PBMC and tumor-infiltrating lymphocytes from melanoma patients (n= 10) and PBMC of normal controls (n= 10) by multicolor flow cytometry. Suppression mediated by sorted ICOS high and ICOS low Treg was assessed in CFSE-based suppression assays with autologous CD4+ CD25− responder cells (RC). Transwell inserts separating Treg from RC were used to evaluate suppression mechanisms used by Treg. ICOS high or ICOS low Treg were coincubated with RC±TCR and IL-2 stimulation. ICOS high and ICOS− Treg were also expanded under conditions previously shown to induce Tr1 from RC. Treg in tumor-infiltrating lymphocytes expressed ICOS (mean fluorescence intensity= 70±10), while Treg in PBMC had low ICOS expression (mean fluorescence intensity= 3.5±2.5, p≤ 0.001). ICOS high Treg up-regulated Treg markers (p≤ 0.0016) and mediated stronger suppression (p≤ 0.001) relative to ICOS low Treg. ICOS high Treg induced Tr1 cells in nonactivated RC and Th2 cells in preactivated RC. ICOS high Treg exposed to Tr1 cytokines expressed IL-10 and suppressed RC (92±12%) in contrast to ICOS low Treg, which mediated low suppression (21±15%; p≤ 0.0028). ICOS high Treg can induce diverse immune responses in RC, depending on activation signals and cytokines present. ICOS high Treg induce Tr1 or Th2 cells depending on the activation state of RC. In a “Tr1” cytokine milieu, ICOS high Treg transit to Tr1.