During immunologic synapse (IS) formation, human CD38 redistributes to the contact area of T cell–antigen-presenting cell (APC) conjugates in an antigen-dependent manner. Confocal microscopy showed that CD38 preferentially accumulated along the contact zone, whereas CD3-ζ redistributed toward the central zone of the IS. APC conjugates with human T cells or B cells transiently expressing CD38–green fluorescent protein revealed the presence of 2 distinct pools of CD38, one localized at the cell membrane and the other in recycling endosomes. Both pools were recruited to the T/APC contact sites and required antigen-pulsed APCs. The process appeared more efficient in T cells than in APCs. CD38 was actively recruited at the IS of T cells by means of Lck-mediated signals. Overexpression of CD38 in T cells increased the levels of antigen-induced intracellular calcium release. Opposite results were obtained by down-regulating surface CD38 expression by means of CD38 siRNA. CD38 blockade in influenza HA-specific T cells inhibited IL-2 and IFN-γ production, PKCθ phosphorylation at Thr538, and PKCθ recruitment to the IS induced by antigen-pulsed APCs. These results reveal a new role for CD38 in modulating antigen-mediated T-cell responses during IS formation.