Toward obtaining a more comprehensive understanding of factors governing activation and/or function during visceral leishmaniasis (VL), we have compared active disease (pre-treatment) versus post-chemotherapy immune response in VL patients by means of ex vivo staining with different cell markers. Our results show that during active disease, the frequency of T cells positive for CD25, CTLA-4 and CD45RO was significantly lower in VL patients compared with healthy controls, whereas cells staining positive for Annexin V and CD95 were significantly higher. In all cases, chemotherapy was able to restore these frequencies to normal levels. Interestingly, significant differences in the frequency of CD18 and in the frequency of CD45RO-positive cells were observed in the CD81 T cell subset. These two frequencies were also significantly higher in bone marrow when compared with peripheral blood, suggesting a possible compartmentalization of certain CD81 T cell populations during active disease. Given that CD81 T cells have been shown to play an essential role in immunity to infection with Leishmania, our data indicate that the lower frequency of CD181 and CD45RO1 lymphocytes in the bone marrow CD81 T cell subset may be considered a biomarker of acute VL.