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Background: Mycosis fungoides (MF) is the most common variant of cutaneous T-cell lymphomas (CTCL). Itching can be a major symptom for patients with CTCL, however, itching associated with MF is not relieved by conventional therapy using anti-histamines, suggesting that histamine is not the main pruritogen. Therefore, the underlying mechanisms of itching in MF patients remain unclear. Objectives: To investigate the clinical and histopathological features associated with MF-related itching. Materials and Methods: Skin sections from MF patients and healthy subjects were used for pathophysiological analysis and evaluation of protease activity. These results were compared with the degree of itching. Results: Of the MF patients, 40% did not report itching and 60% reported itching (moderate itching: 40%; strong itching: 20%). The number of eosinophils, but not mast cells, that infiltrated into skin was increased in the group with strong itching. In the skin of patients, both serine protease activity and immunoreactivity to kallikrein 5 (KLK5), a known itch mediator, increased relative to the grade of itching. Conclusion: These results suggest that KLK5 and eosinophil infiltration may be involved in itching in patients with MF.