The cell adhesion regulating extracellular matrix glycoprotein, thrombospondin (TSP), causes a loss of focal adhesion plaques from spread endothelial cells and fibroblasts. To localize the site on TSP that has focal adhesion-labilizing activity, we initially tested proteolytic fragments of TSP for activity. The heparin-binding fragment has significant focal adhesion-labilizing activity, whereas the nonheparin-binding 140-kDa fragment had no significant activity. These results were consistent with previous data that showed that both a monoclonal antibody to the heparin-binding domain of TSP (A2.5) and heparin neutralized TSP activity. Peptides from putative heparin binding sequences of the amino-terminal heparin-binding domain of TSP were synthesized and tested for their ability to cause loss of focal adhesions. The hep I peptide (amino acids 17-35) caused maximal loss of focal adhesions and was active at 0.1 μM, whereas peptide hep II (74-95) and peptide hep III (170-189) were inactive. The activity of the hep I peptide was neutralized by the addition of heparin and heparan sulfate but not by chondroitin sulfate. The basic amino acids in the hep I sequence appear to be required for focal adhesion-labilizing activity, because modification of the lysine residues at amino acids 24 and 32 rendered the peptide completely inactive. In addition, a peptide from the analogous sequence of mouse TSP 2, in which basic residues are conserved, was nearly as active as hep I from TSP1. These data show that the anti-adhesive activity of TSP is conserved in both TSP1 and TSP2 and that the active site is located in a 19-amino acid sequence in the heparin-binding domain of TSPs.