Vascular permeability factor, or vascular endothelial growth factor (VPF/VEGF) is an important factor in the regulation of vascular growth and vascular permeability. VPF is a secreted, dimeric protein and has 8 cysteine residues conserved with platelet-derived growth factor (PDGF). To study the role of some of these cysteine residues in maintaining the structure and function of VPF, we replaced the codons for the second, third, fourth, and fifth cysteine by serine codons, and expressed the mutant proteins in a mammalian expression system. Cysteine residues 2 and 4 in VPF were found to be directly involved in anti-parallel interchain disulfide bonds, as in PDGF. VPF mutants lacking one of these cysteins were severely impaired in their S-linked dimerization, while upon coexpression of both mutants the ability to form dimers was restored. The VPF mutants lacking cysteine residue 2 or 4 also competed poorly for receptor binding of labeled VPF and had low biological activity, but these defects were also complemented by coexpressing the two mutants, indicating that for efficient receptor binding and activation VPF needs to be a covalent dimer, unlike PDGF-BB. Furthermore, cysteine residue 5 was found to be essential for VPF dimerization and activity, while the mutant lacking cysteine residue 3 was only mildly affected in its ability to dimerize and had partial biological activity.