Expression of uPA is closely related to keratinocyte proliferation. We have demonstrated that 1) only proliferating keratinocytes produce uPA in culture, 2) production and secretion of uPA is regulated during the cell cycle, 3) synthetic growth factor domain (GFD) peptides derived from the EGF-like domain of uPA can stimulate 3 H-thymidine incorporation. In further studies of the growth mechanism of uPA we report that stimulation by GFD peptides is inhibited by the addition of heparin or by heparitinase treatment. This suggests that cell surface heparan sulfate proteo-glycans are involved in uPA mediated growth signal transduction. To investigate the role of syndecans in this process, we purified these 3 5 SO 4 -labeled proteoglycans from human keratinocytes with the polyclonal antibody MS-1-C directed against the cytoplasmic domain of mouse syndecan-1 (Baciu et al., 1994; JBC 269: 696). The radioloabeled syndecans were reacted with uPA, the amino terminal fragment (ATF) and the proteinase domain of uPA that were blotted onto a nitrocellulose membrane. The ATF, but neither uPA nor the proteinase domain of uPA, bound syndecans in a dose dependent manner. The selective affinity of syndecans for ATF was confirmed using recombinant ATF (rATF) and uPA affinity columns. rATF, expressed as a glutathione transferase (GST) fusion protein, was adsorbed onto a glutathione-Sepharose column. When syndecan samples were applied to the rATF column, 20 to 26% of the total radioactivity was absorbed. This material could be eluted with 0.5 M NaCl. When syndecan samples were applied to the rATF column in the presence of 10 μg/ml of heparin all the radioactivity was found in the flow-through fraction. When syndecan samples were applied to the uPA column, only approximately 5% of the total radioactivity was adsorbed. These results clearly demonstrate that ATF can bind syndecans and that these cell surface heparan sulfate proteoglycans may play a role in growth stimulation of keratinocytes.