The iron-binding protein lactoferrin (Lf) is a constituent of neutrophil secondary granules and is discharged into the surrounding medium when neutrophils are activated. Lf released from neutrophils phagocytosing opsonized particles inhibits proliferation of mixed lymphocyte cultures (MLC) and has also been shown to inhibit granulopoiesis, suppress antibody production, and regulate natural killer cell activity. All of these processes are controlled by cytokines, suggesting that Lf may modulate immune responses by inhibiting cytokine activity. When MLC were cultured in round-bottomed wells to crowd the cells together, Lf, 50% saturated with iron, inhibited both proliferation and interleukin-2 (IL-2) release into the supernatants. Inhibition was concentration-dependent and lost at concentrations of Lf greater than 10(-12) mol/L. Lf at 10(-10) mol/L inhibited release of tumor necrosis factor-alpha (TNF) and interleukin- 1 beta (IL-1) into MLC supernatants, as well as inhibiting IL-2 release. TNF in the supernatant was significantly reduced at 5 and 24 hours, becoming less and losing significance by 72 hours. IL-1 in the supernatant was not significantly reduced at 5 and 24 hours, becoming significant at 48 and 72 hours. IL-2 was significantly reduced at 48 and 72 hours and followed the same time course as proliferation. Inhibition was blocked by specific antiserum to Lf, but not by a preimmune serum. Lf, 10(-10) mol/L, also inhibited the production of TNF (49.15% +/- 7.98%; n = 10, P = .032) and IL-1 (42.67% +/- 6.72%; n = 6, P = .032) from endotoxin-stimulated mononuclear cells. As with MLC, inhibition was dose-dependent and abrogated by specific antiserum. Lf did not block the biological action of TNF, IL-1, or IL-2 in specific assays using cytokine-sensitive cell lines. These data suggest that Lf, released from activated neutrophils, acts as a negative feedback mechanism to prevent recruitment and activation of leukocytes in sites of inflammation.