A high number of Leishmania-responder T cells is found in cutaneous leishmaniasis lesions, suggesting that important immunological events occur at the site of infection. Although activated, cytotoxic and regulatory T cells infiltrating into lesions may influence disease pathogenesis, the role of the T cell differentiation pattern of lymphocytes in lesions is unknown. Our aim was to investigate whether the phase of lesion development (early or late) is influenced by the functional status of cells present in inflammatory infiltrate. Activation, cytotoxity and T cell differentiation molecules were evaluated in lesion mononuclear cells by flow cytometry. The frequency of T cells was correlated with the lesion area (r50 68; P50 020). CD41CD251 T cells predominated over CD41CD691 T cells in early lesions (less than 30 days), whereas late lesions (more than 60 days) exhibited more CD41CD691 T cells than CD41CD251 T cells. The duration of illness was correlated positively with CD41CD691 (r50 68; P50 005) and negatively with CD41CD251 T cells (r520 45; P50 046). Most CD81 T cells expressed cytotoxic-associated molecules (CD2441), and the percentages were correlated with the lesion area (r50 52; P50 04). Both CD41 and CD81 effector memory T cells (TEM-CD45RO1CCR7–) predominated in CL lesions and were significantly higher than central memory (TCMCD45RO1CCR71) or naive T cells (CD45RO–CCR71). An enrichment of TEM cells and contraction of naive T cells were observed in lesions in comparison to blood (P50 006) for both CD41 and CD81 T cells. Lesion chronicity is associated with a shift in activation phenotype. The enrichment of TEM and activated cytotoxic cells can contribute to immune-mediated tissue damage.