Osteopontin has been shown to inhibit the induction of inducible nitric oxide synthase (iNOS, or NOS2) by lipopolysaccharide and interferon-γ in the RAW264.7 mouse monocyte/macrophage line and in primary mouse proximal tubule epithelial cells. However, the RAW264.7 cells become refractory to the action of OPN after several subcultures or under dilute culture conditions, possibly because of changes in the composition of the extracellular matrix. We make this suggestion because if the cells are plated on a collagen type I or collagen type IV substrate the inhibitory action of OPN is completely suppressed; this is not the case on substrates consisting of laminin, fibronectin, poly-D-lysine, or poly-(2-hydroxyethylmethylacrylate). These observations imply that macrophages are sensitive to regulation by OPN only in certain physiological contexts. Both hyaluronate, which binds CD44, and rat IgGs are also able to inhibit the induction of NO synthesis by the inflammatory mediators. The similar actions of HA and OPN are consistent with the possibility that CD44 may be a receptor for OPN.