Arg-Gly-Asp (RGD)-containing peptide is a ligand for integrin alpha (v)beta (3) and acts as an angiogenic inhibitor. A novel cyclic RGD peptide, cyclo(-RGDf=V-) (f=V), was synthesized and its biological activities were characterized and compared with its analogs, cyclo(-RGDfV-) (f=V) and cyclo(-RGDf-MeV-) (fMeV). It bound to integrin alpha (v)beta (3) with almost the same affinity as the fV and fMeV analogs. All three compounds inhibited the adhesion and growth of HUVEC cells in a dose-dependent manner in vitro. Out of three, fMeV had the strongest effect, f=V was almost as strong as fMeV, and fV had the least effect. However, in vivo, f=V significantly decreased the intratumoral microvessel density (MVD) in the DLD-1 (human colon cancer cell) inoculated mice, while fMeV had little effect. These results suggest the potential usefulness of the cyclo(-RGDf=V-) as an antiangiogenic agent for clinical use in the future. (C) 2001 Academic Press.