The structural basis for the distinction of viral RNA from abundant self RNA in the cytoplasm of virally infected cells is largely unknown. We demonstrated that the 5′-triphosphate end of RNA generated by viral polymerases is responsible for retinoic acid–inducible protein I (RIG-I)–mediated detection of RNA molecules. Detection of 5′-triphosphate RNA is abrogated by capping of the 5′-triphosphate end or by nucleoside modification of RNA, both occurring during posttranscriptional RNA processing in eukaryotes. Genomic RNA prepared from a negative-strand RNA virus and RNA prepared from virus-infected cells (but not from noninfected cells) triggered a potent interferon-α response in a phosphatase-sensitive manner. 5′-triphosphate RNA directly binds to RIG-I. Thus, uncapped 5′-triphosphate RNA (now termed 3pRNA) present in viruses known to be recognized by RIG-I, but absent in viruses known to be detected by MDA-5 such as the picornaviruses, serves as the molecular signature for the detection of viral infection by RIG-I.