The inhibition of integrin function is a major challenge in medicinal chemistry. Potent ligands are currently in different stages of clinical trials for the antiangiogenic therapy of cancer and age-related macula degeneration (AMD). The subtype alpha5beta1 has recently been drawn into the focus of research because of its genuine role in angiogenesis. In our previous work we could demonstrate that the lack of structural information about the receptor could be overcome by a homology model based on the X-ray structure of the alphavbeta3 integrin subtype and the sequence similarities between both receptors. In this work, we describe the rational design and synthesis of high affinity alpha5beta1 binders, and the optimisation of selectivity against alphavbeta3 by means of extensive SAR studies and docking experiments. A first series of compounds based on the tyrosine scaffold resulted in affinities in the low and even subnanomolar range and selectivities of 400-fold against alphavbeta3. The insights about the structure-activity relationship gained from tyrosine-based ligands could be successfully transferred to ligands that bear an aza-glycine scaffold to yield alpha5beta1 ligands with affinities of approximately 1 nm and selectivities that exceed 10(4)-fold. The ligands have already been successfully employed as selective alpha5beta1 ligands in biological research and might serve as lead structures for antiangiogenic cancer therapy.