To determine whether human milk contained soluble receptors and cytokine antagonists that might contribute to its anti-inflammatory properties, ELISA and enzyme-amplified sensitivity immunoassay methods were used to quantitate soluble intercellular and vascular cell adhesion molecules, soluble E-selectin, soluble IL-6 receptor, IL-1 receptor antagonist, and soluble tumor necrosis factor-α (TNF-α) receptors I and II in human milk and colostrum. Soluble adhesion receptors (soluble intercellular and vascular cell adhesion molecules and soluble E-selectin) were present in colostrum at levels approximately equal to serum, whereas milk levels were significantly lower. Both colostrum and milk contained soluble IL-6 receptor, but the levels present were significantly lower than that reported for serum. The colostrum contents of IL-1 receptor antagonist (672 ± 202 pg/mL), TNF-α receptor I (>3703 ± 305 pg/mL), and TNF-α receptor II(>4507 ± 770 pg/mL) were significantly elevated over serum/plasma levels. Milk levels of IL-1 receptor antagonist and TNF-α receptor I were also greater than serum/plasma levels, but lower than colostrum levels. Examination of sequential milk specimens collected from seven women over a period of 2-6 mo showed that IL-1 receptor antagonist and TNF-α receptors I and II persisted throughout lactation. Column chromatographic fractionation of colostrum and milk demonstrated that soluble TNF-α receptors I and II had molecular sizes up to 60 kD, suggesting that they might be associated with other molecules. Antigen assays for TNF-α in colostrum and milk, as well as chromatographic fractionation experiments, showed that, although present, most TNF-α was not “free” in colostrum or milk, consistent with the observed content of soluble TNF-α receptors I and II. These studies demonstrate that human milk and colostrum contain soluble receptors and cytokine antagonists, materials which could contribute to their anti-inflammatory properties.Abbreviations: sICAM-1,soluble intercellular adhesion molecule-1;sVCAM-1,soluble vascular cell adhesion molecule-1;sE-selectin,soluble E-selectin;sIL-6R,soluble IL-6 receptor;TNF,tumor necrosis factor;sTNF RI,soluble tumor necrosis factor-α receptor I;sTNF RII,soluble tumor necrosis factor-α receptor II;IL-1RA,IL-1 receptor antagonist;EASIA,enzyme-amplified sensitivity immunoassay;HRP,horseradish peroxidase