During an innate immune response, macrophages recognize viruses by their pattern recognition receptors. In this study, we have studied the role of membrane-associated TLRs and cytoplasmic retinoic acid inducible gene-I (RIG-I)-like receptors (RLR) in regulation of IFN-beta, IL-29, IL-1beta, and IL-18 production and caspases 1 and 3 activation in human macrophages. We provide evidence that TLRs are mainly involved in transcriptional up-regulation of IL-1beta gene expression, whereas cytosolic dsRNA recognition pathway stimulates powerful IFN-beta and IL-29 gene transcription. However, robust IL-1beta secretion occurred only if two TLRs were triggered simultaneously or if a single TLR was activated in conjunction with the RLR pathway. Markedly, TLR activation did not stimulate IL-18 processing or secretion. In contrast, triggering of cytosolic RNA recognition pathway with poly(I:C) transfection or influenza A virus infection resulted in caspase-1- and -3-mediated proteolytic processing of pro-IL-18 and secretion of biologically active IL-18. Furthermore, caspase 3-dependent processing of pro-IL-18 was also observed in human HaCaT keratinocytes, and forced expression of RIG-I and its downstream effector, mitochondrial antiviral signaling protein, activated proteolytic processing of pro-IL-18, caspase-3, and apoptosis in these cells. The present results indicate that in addition to robust IFN-beta, IL-29, IL-1beta, and IL-18 generation, RIG-I/mitochondrial antiviral signaling protein pathway activates caspase-3, suggesting a role for these RIG-I-like receptors beyond the innate cytokine response, hence, in the induction of apoptosis of the virus-infected cell.