Several observations suggest endogenous suppressors of inflammatory mediators are present in human blood. α-1-Antitrypsin (AAT) is the most abundant serine protease inhibitor in blood, and AAT possesses anti-inflammatory activity in vitro and in vivo. Here, we show that in vitro stimulation of whole blood from persons with a genetic AAT deficiency resulted in enhanced cytokine production compared with blood from healthy subjects. Using whole blood from healthy subjects, dilution of blood with RPMI tissue-culture medium, followed by incubation for 18 h, increased spontaneous production of IL-8, TNF-α, IL-1β, and IL-1R antagonist (IL-1Ra) significantly, compared with undiluted blood. Dilution-induced cytokine production suggested the presence of one or more circulating inhibitors of cytokine synthesis present in blood. Serially diluting blood with tissue-culture medium in the presence of cytokine stimulation with heat-killed Staphylococcus epidermidis (S. epi) resulted in 1.2- to 55-fold increases in cytokine production compared with S. epi stimulation alone. Diluting blood with autologous plasma did not increase the production of IL-8, TNF-α, IL-1β, or IL-1Ra, suggesting that the endogenous, inhibitory activity of blood resided in plasma. In whole blood, diluted and stimulated with S. epi, exogenous AAT inhibited IL-8, IL-6, TNF-α, and IL-1β significantly but did not suppress induction of the anti-inflammatory cytokines IL-1Ra and IL-10. These ex vivo and in vitro observations suggest that endogenous AAT in blood contributes to the suppression of proinflammatory cytokine synthesis.