Hepatocyte growth factor/scatter factor (HGF/SF) is a heparan sulfate (HS)-binding growth factor and morphogen for mammary epithelial cells that is produced by mammary stromal fibroblasts. HS chains, purified as peptidoglycans from a panel of cell lines representative of the ductal epithelial cell (Huma 123), the myoepithelial cell (Huma 109), the stromal fibroblast (Rama 27), and malignant mammary epithelial cells (MCF-7 and ZR-75), were used in a biosensor-based assay to identify the classes of HGF/SF-binding sites in the polysaccharide chains. At least three distinct binding sites were identified. One site exhibits fast association and fast dissociation kinetics [kass (1.4-7.7) x 10(6) M-1 s-1; kdiss 0. 0032-0.0096 s-1] and is present on the HS from benign Huma 123 epithelial cells, Huma 109 myoepithelial-like cells, and ZR-75 malignant cells. The second binding site, found on HS from the malignant MCF-7 cells, has slower HGF/SF-binding kinetics (kass 0.20 x 10(6) M-1 s-1; kdiss 0.00055 s-1). The third binding site possesses fast association and slow dissociation kinetics (kass 1.1 x 10(6) M-1 s-1; kdiss 0.00020 s-1) and was found on the HS isolated from the culture medium of the Huma 123 benign epithelial cells. The first and second binding sites have a similar Kd, 1-3 nM, while the third binding site has a considerably higher affinity for HGF/SF (Kd 200 pM). The three binding sites seem to be mutually exclusive, since each sample of HS possessed just one of the sites.