The membrane glycoprotein CD4 is required for optimal antigen-mediated activation of CD4+ T cells restricted by class II molecules of the major histocompatibility complex (MHC). CD4 cross-linking by anti-CD4 antibodies or binding by human immunodeficiency virus (HIV) gp120 has been shown to inhibit antigen-dependent and -independent T cell activation, abrogating T cell proliferation, IL-2 synthesis and the increase in the intracellular calcium concentration. The molecular basis of these opposing phenomena is ill-defined. To characterize further the inhibitory role of the CD4 molecule, we investigated the effects of CD4 ligands on the transcription factors regulating the IL-2 gene enhancer and IL-2 synthesis. We first confirmed that pre-treatment of peripheral human CD4+ T lymphocytes by CD4 ligands, HIV gp120 or anti-CD4 monoclonal antibodies inhibited IL-2 production and cell proliferation, which was normally induced by an anti-CD3 antibody (UCHT1) plus a protein kinase C activator (PMA). Moreover, these CD4 ligands inhibited the proliferation and synthesis of IL-2 induced by activators bypassing membrane events, i.e. PMA and calcium ionophore, pointing to an active signaling pathway triggered by the CD4 molecule. Gp120 and anti-CD4 antibodies induced a specific, significant decrease in the binding activity of NF-AT, NF-kappa B and AP-1, three transcription factors regulating IL-2 gene enhancer activity, as demonstrated by electrophoretic mobility shift assays. Inhibition was similarly observed following cell activation by activators involving membrane events and those bypassing them. These results strongly suggest that the inhibition mediated by cross-linking of the CD4 molecule is at least partly due to negative signal down-regulating the availability of nuclear factors necessary for the regulation of IL-2 gene transcription.