ADP-ribosyltransferase (ADPRT) is a glycosylphosphatidylinositol-anchored cell surface enzyme on CTL. Expression of this enzyme correlates with suppression of CTL functions in the presence of its substrate beta-nicotinamide adenine dinucleotide (NAD). To investigate the immunoregulatory importance of ADPRT on normal lymphocytes in vivo, NAD was injected into mice and the effects on cell-mediated and humoral immunity were assessed. Induction of both delayed-type hypersensitivity and CTL, but not Ab responses, are shown to be suppressed by NAD. Consistent with this, mature T cells, but not B cells or macrophages, express ADPRT and are able to ADP-ribosylate cell surface proteins. ADP-ribosylated molecules were identified as LFA-1, CD8, CD27, CD43, CD44, and CD45. Concomitant to ADP-ribosylation of these molecules, T cell trafficking to secondary lymphoid organs is suppressed by NAD. To examine whether this is due to effects of NAD on cell activation, Ag-stimulated responses were assayed in vitro. NAD is shown to inhibit induction of cell proliferation, cytotoxicity, and cytokine secretion. It is suggested that ADPRT regulates T cells on the level of transmembrane signaling via ADP-ribosylation of cell surface molecules. This effect is reported to be indirect, as it involves transmission of signals through TCRs, which are not ADP-ribosylated.