Although elevated CD4(+)Foxp3(+) regulatory T cell (Treg) frequencies within tumors are well documented, the functional and phenotypic characteristics of CD4(+)Foxp3(+) and CD4(+)Foxp3(-) T cell subsets from matched blood, healthy colon, and colorectal cancer require in-depth investigation. Flow cytometry revealed that the majority of intratumoral CD4(+)Foxp3(+) T cells (Tregs) were Helios(+) and expressed higher levels of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and CD39 than Tregs from colon and blood. Moreover, ∼30% of intratumoral CD4(+)Foxp3(-) T cells expressed markers associated with regulatory functions, including latency-associated peptide (LAP), lymphocyte activation gene-3 (LAG-3), and CD25. This unique population of cells produced interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), and was ∼50-fold more suppressive than Foxp3(+) Tregs. Thus, intratumoral Tregs are diverse, posing multiple obstacles to immunotherapeutic intervention in colorectal malignancies.Mucosal Immunology advance online publication, 25 September 2013; doi:10.1038/mi.2013.62.