<title>Abstract</title>Detailed analysis of the cells that infiltrate lesional skin cannot be performed in skin biopsy specimens using immunohistochemistry or cell separation techniques because enzyme treatments applied during the isolation step can destroy small amounts of protein and minor cell populations in the biopsy specimen. Here, we describe a method for isolating T cells from drops of whole blood obtained from lesions during skin biopsy in patients with cutaneous T-cell lymphoma. Lesional blood is assumed to contain lesional resident cells, cells from capillary vessels, and blood overflowing from capillary vessels into the lesion area. The lesional blood showed substantial increases in distinct cell populations, chemokines, and the expression of various genes. The proportion of CD8⁺CD45RO⁺ T cells in the lesional blood negatively correlated with the modified severity-weighted assessment tool scores. CD4⁺CD45RO⁺ T cells in the lesional blood expressed genes associated with the development of cancer and progression of cutaneous T-cell lymphoma. In addition, CD8⁺CD45RO⁺ T cells in lesional blood had unique T-cell receptor repertoires in lesions of each stage. Assessment of lesional blood drops might provide new insight into the pathogenesis of mycosis fungoides and facilitate evaluation of the treatment efficacy for mycosis fungoides as well as other skin inflammatory diseases.