We have previously reported that the hyaluronan (HA) receptor RHAMM (Receptor for HA Mediated Motility) [Turley et al., 1991] contains two HA binding motifs located within a 35 amino acid region of its C-terminus end [Yang et al., 1993] and that HA stimulation of the motility of ras-transformed fibroblasts is mediated via its interaction with RHAMM. Here we show that RHAMM also contains binding sites for heparin (HP) and that interaction of HP with these sites can regulate the locomotion of ras-transformed fibroblasts. At low concentrations (0.01 mg/ml), HP inhibited HA-induced locomotion of ras-transformed cells in a manner independent of RHAMM. At higher, but still physiological concentrations (0.1 mg/ml), HP alone stimulated cell locomotion and this stimulation appeared to be RHAMM-dependent as it was blocked by anti-RHAMM antibodies. Other related glycosaminoglycans such as chondroitin sulfate and dermatin sulfate had no effect on cell motility. In ligand blotting assays, GST-RHAMM fusion protein was shown to bind biotin-labelled HP and this binding was displaceable with unlabelled HP. In similar ligand binding analyses conducted with truncations of RHAMM fusion protein, the HP binding region was found to be localized in the same 35 amino acid segment of RHAMM that contains the two HA binding domains. Synthetic peptides corresponding to these HA binding domains were retained on and bound effectively to an HP-Sepharose affinity column. Fusion proteins generated by linkage of these peptides to the non-HP binding amino terminus of RHAMM conferred HP binding capacity to the genetically engineered proteins. Conversely, deletion of the HA binding domains of RHAMM resulted in fusion proteins devoid of HP binding activity. The relative affinities of RHAMM for HA and HP, as determined by competition and transblot assays as well as quantification of binding at various salt concentrations, indicated that RHAMM had lower affinity for HP than that for HA. These results demonstrate the existence of a new HP binding motif that has biological relevance to cell locomotion.