We have identified a group of DNA molecules that bind to platelet-derived growth factor (PDGF)-AB with subnanomolar affinity from a randomized DNA library using in vitro selection. Individual ligands cloned from the affinity-enriched pool bind to PDGF-AB and PDGF-BB with comparably high affinity (K_d ≈ 10^-10 M) and to PDGF-AA with lower affinity (>10^-8 M), indicating specific recognition of the PDGF B-chain in the context of the hetero- or homodimer. The consensus secondary structure motif for most of the high-affinity ligands is a three-way helix junction with a three-nucleotide loop at the branch point. Photo-cross-linking experiments with 5-iodo-2‘-deoxyuridine-substituted ligands establish a point contact between a thymidine nucleotide in the helix junction loop region and phenylalanine 84 of the PDGF-B chain. Representative minimal DNA ligands inhibit the binding of ¹²⁵I-PDGF-BB but not of ¹²⁵I-PDGF-AA to PDGF α- or β-receptors expressed in porcine aortic endothelial (PAE) cells in a concentration-dependent manner with half-maximal effects of ≈1 nM. The same ligands also exhibit a similar inhibitory effect on PDGF-BB-dependent [³H]thymidine incorporation in PAE cells expressing the PDGF β-receptors. These DNA ligands represent a novel class of specific and potent antagonists of PDGF-BB and, by inference, PDGF-AB.