We have constructed synthetic peptides modeled on the sequences of (i) Arg-Gly-Asp, present in fibrinogen, fibronectin, and von Willebrand factor, and of (ii) the fibrinogen gamma chain (gamma 400-411) His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val. The concentration of each peptide that inhibits 50% of 125I-labeled fibrinogen binding to thrombin-stimulated platelets (IC50) was then determined. The IC50 for (gamma 400-411) was 48-180 microM at a fibrinogen concentration of 60 micrograms/ml. A substitution of arginine for alanine at position 9 decreased the IC50 to 14.5 microM. Arginine substitutions for all other residues on the amino-terminal side of the peptide Arg9-Gly-Asp-Val resulted in an IC50 of 0.4-0.8 microM, and the IC50 of the peptide Arg13-Gly-Asp-Val was 0.2-0.3 microM. This contrasts with an IC50 of 200 microM for Arg5-Gly-Asp-Val-Arg4 and an IC50 greater than 1 mM for the peptide Arg12. The inhibitory effect resulted primarily in a decreased affinity of fibrinogen binding to platelets, although the number of available binding sites had also decreased. Binding was completely inhibited. At concentrations between 10 and 18 microM, Arg9-Gly-Asp-Val blocked all ADP-induced aggregation in citrated platelet-rich plasma. The peptide Tyr-His-His-Lys-Arg-Lys-Arg-Lys-Gln-Arg-Gly-Asp-Val was labeled with 125I to quantitate its binding to thrombin-stimulated platelets; at saturation, 59,990 molecules were bound per cell (Kd = 3.8 X 10(-7) M). These modified synthetic peptides bind to platelets with the same affinity as does intact fibrinogen and inhibit platelet function. The increased affinity of these modified peptides is greater than 20-fold that of peptides comprised of only native sequences and is a prerequisite for the potential antithrombotic use of these agents.