A major obstacle hampering the therapeutic application of regulatory T (Treg) cells is the lack of suitable extracellular markers, which complicates their identification/isolation. Treg cells are normally isolated via CD25 (IL‐2Rα) targeting, but this protein is also expressed by activated CD4⁺ effector T (Teff) lymphocytes. Other extracellular (positive or negative) Treg selection markers (e.g., HLA‐DR, CD127) are also nonspecific. CD26 is an extracellular peptidase whose high expression has been traditionally used as an indicator of immune activation and effector functions in T cells. Now, we provide flow cytometry data showing high levels of CD26 within CD4⁺CD25⁻ or CD4⁺FoxP3^−/low effector T (Teff) lymphocytes, but negative or low levels (CD26^−/low) in Treg cells selected according to the CD4⁺CD25^high or the CD4⁺FoxP3^high phenotype. Unlike the negative marker CD127 (IL‐7Rα), which is down modulated in CD4⁺ Teff lymphocytes after TCR triggering, most of these cells upregulate CD26 and take a CD4⁺CD25^+/highCD26⁺ phenotype upon activation. In contrast, there is only a slight upregulation within Treg cells (CD4⁺CD25^highCD26^−/low). Thus, differences in CD26 levels between Treg and Teff subsets are stable, and assessment of this marker, in combination with others like CD25, FoxP3, or CD127, may be useful during the quantitative evaluation or the isolation of Treg cells in samples containing activated Teff lymphocytes (e.g., from patients with autoimmune/inflammatory diseases). © 2012 International Society for Advancement of Cytometry