Activated platelets are known to modulate immune responses by secreting or shedding a range of immunomodulatory substances. We examined the influence of activated platelets on cytokine production by normal human mononuclear cells, induced by tetanus toxoid (TT), human thyroglobulin (TG), Escherichia coli LPS, or intact Porphyromonas gingivalis. Addition of platelets activated by thrombin-receptor-activating peptide enhanced IL-10 production induced by LPS (p < 0.001), TG (p < 0.05), and P. gingivalis (p < 0.01), and reduced the production of TNF-α induced by LPS (p < 0.001), TG (p < 0.05), and P. gingivalis (p < 0.001), and of IL-6 in LPS- and P. gingivalis-stimulated cultures (p < 0.001). Similar effects on IL-10 and TNF-α production were observed on addition of platelet supernatant to mononuclear cells, whereas addition of recombinant soluble CD40L mimicked the effects on IL-10 production. Moreover, Ab-mediated blockade of CD40L counteracted the effect of platelets and platelet supernatants on TNF-α production. Monocytes separated into two populations with respect to IL-10 production induced by TG; the high-secreting fraction increased from 0.8 to 2.1% (p < 0.001) on addition of activated platelets. Adherence of platelets increased TG- and TT-induced IL-10 secretion by monocytes (p < 0.05). In addition, activated platelets inhibited CD4(+) T cell proliferation elicited by TT (p < 0.001) and P. gingivalis (p < 0.001). Our findings suggest that activated platelets have anti-inflammatory properties related to the interaction between CD40L and CD40, and exert a hitherto undescribed immunoregulatory action by enhancing IL-10 production and inhibiting TNF-α production by monocytes.