It has recently become apparent that certain small interfering RNA (siRNA) sequences stimulate the innate immunity through endosomal Toll-like receptors (TLR), particularly TLR7 and TLR8. However, it remains unclear whether siRNA duplexes act as specific ligands for these receptors. To address this question and to overcome the problem of immune activation by siRNA, several RNA sequences were chemically synthesized and their effects were investigated. Results indicate that human peripheral blood mononuclear cells (PBMC) recognize and respond to a large number of sense or antisense single-stranded (ss) siRNA. In most cases immunostimulatory RNA motifs are more effectively recognized by innate immunity in the context of ss siRNA as compared to siRNA duplexes. Novel immunostimulatory RNA motifs were identified and their replacement with adenosines abrogated immune activation. Most notably, replacement of the 2′-hydroxyl uridines with either 2′-fluoro, 2′-deoxy or 2′-O-methyl uridines abrogated immune activation. Thus, immune recognition of RNA by TLR can be evaded by 2′-ribose modifications of only uridines. Collectively, the data should facilitate the development of siRNA therapeutics and expand the understanding of how RNA is sensed by innate immunity.