Confluent cultures of human endothelial cells deposit into extracellular matrix (ECM) distinct heparan sulfate proteoglycans (HSPG) which modulate acidic fibroblast growth factor's (aFGF) ability to stimulate human endothelial cell mitogenic capacity. Extracellular matrix 'IS-HSPG were isolated from cultures metabolically labelled with Na235S04 by DEAE-Sepharose, Sepharose CL-4B, and aFGF-Affi-Gel 15 column chromatography and identified by resistance to chondroitinase ABC and sensitivity to nitrous acid. Fifty to sixty percent of the 3'S-HSPG deposited into ECM do not bind aFGF. The bound 35S-HSGP (40-50% of the total counts applied) eluted from the aFGF-Affi-Gel column after the addition of buffer containing 2 M NaCI. aFGF-binding and aFGF-nonbinding 'IS-HSPG were individually pooled and further purified by Sepharose CL-4B column chromatography. 35S-HSPG which bind aFGF, designated HSPG', were 100-fold superior to heparin in augmenting the mitogenic efficacy of aFGF in sparse proliferating cultures. In contrast, however, "'S-HSPG, which did not bind aFGF, designated HSPG', inhibited aFGF-stimulated proliferation in both sparse and subconfluent endothelial cell cultures. The majority of the biological activity of both aFGF-potentiating HSPG' and aFGF-inhibitory HSPG' was contained in the glycosaminoglycan chains released by alkaline borohydride treatment of intact HSPG" or HSPG', respectively. 3H-Core protein derived from HSPG' or HSPG' contained only minor biological activity. The ability of heparitinase or heparinase (Flavobacterium heparinurn) to abolish biological activity differed, depending upon the HSPG tested, also suggested that these are two distinct HSPGs.