Chemotaxis stimulated by formyl-leucine-methionine-phenylalanine (fMLP), a peptide that is chemotactic for inflammatory cells, has previously been shown to be attenuated by inhibitors of NO synthase. Hyaluronan (HA) is a glycosaminoglycan that regulates cell locomotion through interaction with cell receptors such as RHAMM. Since we have shown that NO stimulation of macrophage chemotaxis requires RHAMM:HA interaction, we hypothesized that fMLP would regulate the expression of both HA and RHAMM in a NO-dependent manner. In order to study the role of NO, RHAMM and HA in fMLP-stimulated chemotaxis, we used a murine macrophage cell line (S1) in a modified Boyden chamber assay. fMLP (10nM) stimulated macrophage chemotaxis as determined by checkerboard analysis. This concentration of fMLP also increased the expression of RHAMM at both the mRNA and protein level, and increased the accumulation of HA in the medium. L-N-arginine methyl ester (5 mM, L-NAME), a blocker of NO synthase, blocked fMLP-stimulated chemotaxis coincident with an inhibition of RHAMM expression. Under these conditions, there was a greater accumulation of HA in the medium suggesting that receptor-mediated internalization of HA is involved in the response to fMLP. Sodium nitroprusside (0.5 nM, SNP), a donor of NO, stimulated chemotaxis and increased RHAMM and HA expression, effects that were not blocked by L-NAME. The HA-binding region of aggrecan and synthetic HA-binding peptides were able to inhibit both fMLP- and SNP-stimulated macrophage chemotaxis, as was an anti-RHAMM antiserum previously shown to interfere with RHAMM:HA interactions. We conclude that fMLP-stimulated chemotaxis requires NO which, in turn, upregulates RHAMM and HA. Subsequent receptor:ligand interaction results in internalization of HA, a requirement for the chemotactic response.