Using the systematic evolution of ligands by exponential enrichment (SELEX) method, we have identified oligonucleotides that bind to human IgE with high affinities and high specificity. These ligands were isolated from three pools of oligonucleotides, each representing 10(15) molecules: two pools contained 2'-NH2 pyrimidine-modified RNA with either 40 or 60 randomized sequence positions, and the third pool contained ssDNA with 40 randomized sequence positions. Based on sequence and structure similarities, these oligonucleotide IgE ligands were grouped into three families: 2'-NH2 RNA group A ligands are represented by the 35-nucleotide truncate IGEL1.2 (Kd = 30 nM); 2'-NH2 RNA group B ligands by the 25-nucleotide truncate IGEL2.2 (Kd = 35 nM); and the ssDNA group ligands by the 37-nucleotide truncate DI 7.4 (Kd = 10nM). Secondary structure analysis suggests G quartets for the 2'-NH2 RNA ligands, whereas the ssDNA ligands appear to form stem-loop structures. Using rat basophilic leukemia cells transfected with the human high-affinity IgE receptor Fc epsilon RI, we demonstrate that ligands IGEL1.2 and D17.4 competitively inhibit the interaction of human IgE with Fc1 epsilon RI. Furthermore, this inhibition is sufficient to dose-dependently block IgE-mediated serotonin release from cells triggered with IgE-specific Ag or anti-IgE Abs. Therefore, these oligonucleotide ligands represent a novel class of IgE inhibitors that may prove useful in the fight against allergic diseases.