CD44 is a cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is involved in processes ranging from leukocyte recruitment to wound healing. In the immune system, the binding of hyaluronan to CD44 is tightly regulated, and exposure of human peripheral blood monocytes to inflammatory stimuli increases CD44 expression and induces hyaluronan binding. Here we sought to understand how mouse macrophages regulate hyaluronan binding upon inflammatory and anti-inflammatory stimuli. Mouse bone marrow-derived macrophages stimulated with tumor necrosis factor ␣ or lipopolysaccharide and interferon-␥ (LPS/IFN␥) induced hyaluronan binding by up-regulating CD44 and down-regulating chondroitin sulfation on CD44. Hyaluronan binding was induced to a lesser extent in interleukin-4 (IL-4)-activated macrophages despite increased CD44 expression, and this was attributable to increased chondroitin sulfation on CD44, as treatment with -D-xyloside to prevent chondroitin sulfate addition significantly enhanced hyaluronan binding. These changes in the chondroitin sulfation of CD44 were associated with changes in mRNA expression of two chondroitin sulfotransferases, CHST3 and CHST7, which were decreased in LPS/IFN␥-stimulated macrophages and increased in IL-4-stimulated macrophages. Thus, inflammatory and anti-inflammatory stimuli differentially regulate the chondroitin sulfation of CD44, which is a dynamic physiological regulator of hyaluronan binding by CD44 in mouse macrophages.Macrophages differentiate from peripheral blood monocytes upon recruitment to the tissues. Under homeostatic conditions this process occurs constitutively to maintain a population of resident macrophages that provide a first line of defense against invading microorganisms (for review, see Refs. 1 and 2). This is of particular importance in some tissues, such as the lungs, where phagocytosis of bacteria by resident alveolar macrophages can be sufficient to clear infection (3). During inflammatory conditions, recruitment of a different subpopulation of monocytes occurs, and the number of macrophages in the tissues is greatly increased. Macrophages initially undergo "classical activation" in response to IFN␥ and toll-like receptor (TLR) 6 ligands such as LPS and display increased anti-microbial abilities due to increased production of reactive nitrogen and oxygen species (for review, see Refs. 4 and 5). The production of proinflammatory and T helper cell 1 (T H 1)-polarizing cytokines by these LPS-and IFN␥-polarized (M1) macrophages affects both the local and systemic immune response. Recruited macrophages are also important for resolving inflammation, as they phagocytose inflammation-inducing agents such as bacteria, cellular debris, and degraded extracellular matrix components (6, 7). Furthermore, increased levels of anti-inflammatory or T helper cell 2 (T H 2)-polarizing cytokines such as IL-4 result in the "alternative activation" of macrophages. These alternatively activated (M2) macrophages produce anti-inflammatory cytokines ...