We have isolated single-chain Fv fragments directed against human endothelial cells from a novel fully synthetic human scFv library (scFv 479). This library was constructed using the variable germline segments DP47 and DPkappa9 as scaffolds. Complementarity determining regions 3 (CDR) of the variable heavy and light chain were introduced with a length of 9 amino acid residues. In total, 16 amino acid positions of all six CDRs exposed in the antigen-binding site were randomized and the library was produced from synthetic oligonucleotides encoding the entire scFv fragment. From this library endothelial-specific scFv fragments were either selected using the recombinant extracellular domain of human endoglin (CD105) or by cell selections with human dermal microvascular endothelial cells (HDMEC). These scFv fragments might be useful for the generation of vascular or tumor targeting agents in cancer therapy.