Macrophage chemotaxis, a critical component of the inflammatory response to lung injury, has previously been shown to be attenuated by inhibitors of NO synthase. Hyaluronan (HA), a glycosaminoglycan that accumulates in the lung during pulmonary inflammation, regulates cell locomotion through interaction with cell receptors such as RHAMM (Receptor for HA-Mediated Motility). Since NO has been implicated in pulmonary damage, we hypothesized that NO would regulate the expression of both HA and RHAMM during stimulation of macrophage chemotaxis. We used a murine macrophage cell line (S1) in a modified Boyden chamber assay. HA stimulated macrophage chemotaxis in a bell-shaped, dose-dependent manner, with maximal effect at 0.1 μg/ml. L-N-arginine methyl ester (L-NAME), a blocker of NO synthase, failed to inhibit HA-stimulated chemotaxis over a large range of concentrations (0.1-5 mM). Sodium nitroprusside (SNP), a NO donor, stimulated macrophage chemotaxis in a dose-dependent manner with maximal effect at 0.5 nM. This concentration of SNP also maximally upregulated the expression of RHAMM and HA. The HA-binding region of aggrecan and synthetic HA-binding peptides inhibited NO-stimulated macrophage chemotaxis, as did an anti-RHAMM antiserum previously shown to interfere with RHAMM:HA interactions. Interestingly, addition of L-NAME to quiescent S1 cells down-regulated RHAMM and increased the media content of HA suggesting a block of receptor-mediated internalization of HA. We conclude that NO regulates the normal homeostasis of the RHAMM:HA axis in quiescent cells, but, at higher concentrations, increases the expression of RHAMM and HA, stimulating macrophage chemotaxis through the interaction of HA with RHAMM. We speculate that regulation of HA content after lung injury may provide a useful means of limiting pulmonary inflammation and subsequent fibrosis.