Vascular endothelial cells, providing a nonthrombogenic surface to the lumenal aspect of blood vessels, are anchored to matrix adhesion molecules in the subendothelium through their respective receptors belonging to a superfamily of integrins. We analyzed the reactivity of synthetic peptide analogs of adhesive proteins toward human umbilical vein endothelial cells (HUVEC), assaying their detachment from extracellular matrix and attachment to extracellular matrix components in vitro. Synthetic peptide analogs Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-Asp-Val (RGDV), Arg-Gly-Asp-Ser (RGDS), and Arg-Gly-Asp-Phe (RGDF), which are analogous to “cell adhesion site” of fibronectin, vitronectin, von Willebrand factor, and alpha-chain of human fibrinogen, respectively, caused significant detachment of HUVEC from the extracellular matrix in vitro at the concentrations ranging from 0.5 to 1.5 mmol/L. They also interfered with attachment of HUVEC to surfaces coated with subendothelial extracellular matrix or its components. The synthetic peptide analog of HHLGGAKQAGDV, which is homologous to the gamma-chain of human fibrinogen sequence 400–411, did not cause any measurable effect on the integrity of HUVEC monolayers (detachment and attachment). “Hybrid” peptides bearing salient features of both sequences, ie, Ala-Lys-Gln-Arg-Gly-Asp-Phe (AKQRGDF) and Lys- Gln-Arg-Gly-Asp-Phe (KQRGDF), had an attenuated effect on the detachment of HUVEC from extracellular matrix. Thus, the integrity of the human endothelial cell monolayer anchored to the extracellular matrix, as measured in detachment and attachment assays, is disturbed by peptides containing RGD sequence whereas the synthetic peptide His- His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) is nonreactive.