We have recently described the cDNA and predicted protein structure of a natural soluble CD4 ligand, IL-16. IL-16 is chemotactic for CD4+ T cells and induces functional IL-2 receptors in CD4+ T cells. The binding of IL-16 to CD4 results in activation of p56(lck), whose adaptor function is essential for the chemotactic response. Subsequently, increases in intracellular Ca2+ and phosphatidylinositol 1,4,5-trisphosphate occur, as does translocation of protein kinase C from cytosol to membrane. Because of the similarities between these signals and functions and those noted for the CD4 ligand HIV-1 gp120, we investigated the potential regulatory effects of IL-16 on CD3/TCR-mediated lymphocyte activation. Preincubation of human T cells with IL-16 up to 24 h before activation with plate-bound anti-CD3 Abs reduced T cell activation by 80%, as monitored by IL-2R expression and [3H]thymidine uptake. If IL-16 was added following anti-CD3 activation, no suppression was noted. The suppressive effects of preincubation with IL-16 were not rescued by the addition of rIL-2 and were not the result of priming for anti-CD3-induced apoptosis. In addition, IL-16 had no effect on surface expression of CD3 or CD4. However, IL-16 did reduce the magnitude of the anti-CD3-induced intracellular Ca2+ increase. These studies indicate that while the interaction of CD4 with its natural ligand, IL-16, results in Ag-independent chemotaxis and IL-2R expression, this pro-inflammatory state is associated with subsequent transient inhibition of responsiveness via the CD3/TCR complex.