The loss of CD26 expression was proposed to be a constant feature of circulating Sézary cells by flow cytometric immunophenotyping (FCIP), but the experience with CD26 is limited. To establish its usefulness, CD26 results were correlated with morphologic, molecular, and immunophenotypic findings. Based on FCIP of 179 samples of peripheral blood, CD26 negativity was found in 59.3% of cases with Sézary syndrome (SS), 33.3% of mycosis fungoides (MF), 14.2% of benign dermatosis (BD), and no control cases. In diagnostic subgroups of SS based on morphologic, molecular, and immunophenotypic criteria, the percentage of CD26- cases varied from 41.1% to 63.6%. The specificity of a CD26- result was inferior to that of T-cell antigen loss in differentiating SS from MF and BD. CD26 offers lower diagnostic performance than previously suggested; however, in addition to the findings of major T-cell antigen loss, it could improve sensitivity of FCIP in patients with SS.