Although prevention of mother-to-child HIV transmission by antiretroviral drugs has been shown to be effective, the short- and long-term effects of treatment are not well known. Several reports suggest that antiretroviral drugs act not only by inhibiting viral replication, but also by improving antiviral immunity. In particular, treatment with nucleoside analogs was found to increase CD40 ligand (CD40L; CD154) levels, an inducible molecule expressed on activated T lymphocytes. The present study investigated potential immunostimulatory effects of antiretroviral treatment (ART) in uninfected HIV-exposed infants, receiving either ante- or postnatal therapy to reduce the transmission rate. To this end, we analyzed CD40L expression in unstimulated lymphocytes from peripheral blood samples of uninfected infants vertically exposed to HIV and subjected to ART. The CD45 PanLeucogating strategy was applied in flow cytometry to analyze the lymphocytes of 41 cases and 64 age-matched infants, taken as control. We found increased CD154 expression on both CD4+ and CD8+ lymphocytes in ART-treated infants, compared with the controls. CD154 was apparently functional, because the expression of CD86 on monocytes was enhanced; moreover, inhibition of the CD40-CD40L interaction produced downmodulation of CD86. From these results, we conclude that CD4+ and CD8+ lymphocytes of HIV-exposed noninfected infants, who have been exposed to antiretroviral drugs in fetal and early life, display enhanced CD154 expression and costimulatory activity.