Imaging of angiogenic processes is of great interest in preclinical research as well as in clinical settings. The most commonly addressed target structure for imaging angiogenesis is the integrinα_vβ₃. Here we describe the synthesis and evaluation of [¹⁸F]FProp-Cys*-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys*-OH, a radiolabelled peptide designed to selectively target the integrinα₅β₁. Conjugation of 4-nitrophenyl-(RS)-2-[¹⁸F]fluoropropionate provided [¹⁸F]FProp-Cys*-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys*-OH in high radiochemical purity (>95%) and a radiochemical yield of approx. 55%. In vitro evaluation showedα₅β₁binding affinity in the nanomolar range, whereas affinity toα_vβ₃andα_IIbβ₃was >50 μM. Cell uptake studies using human melanoma M21 (α_vβ₃-positive andα₅β₁-negative), human melanoma M21-L (α_vβ₃-negative andα₅β₁-negative), and human prostate carcinoma DU145 (α_vβ₃-negative andα₅β₁-positive) confirmed receptor-specific binding. The radiotracer was stable in human serum and showed low protein binding. Biodistribution studies showed tumour uptake ranging from 2.5 to 3.5% ID/g between 30 and 120 min post-injection. However, blocking studies and studies using mice bearingα₅β₁-negative M21 tumours did not confirm receptor-specific uptake of [¹⁸F]FProp-Cys*-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys*-OH, although this radiopeptide revealed high affinity and substantial selectivity toα₅β₁in vitro. Further experiments are needed to study the in vivo metabolism of this peptide and to develop improved radiopeptide candidates suitable for PET imaging ofα₅β₁expression in vivo.