Innate immunity recognizes microbial nucleic acids by endosomal TLR and cytosolic recognition systems. Despite increasing knowledge on the properties of nucleic acid recognition for viral RNA and bacterial DNA, little is known about the immunogenicity of prokaryotic RNA. Here we show that bacterial RNA is a potent trigger for type-I IFN secretion in human PBMC. Activation of human plasmacytoid dendritic cells was dependent on endosomal maturation and could be blocked by a TLR7-specific inhibitor. Murine plasmacytoid dendritic cells from TLR7-deficient mice were unresponsive to bacterial RNA. Surprisingly, in myeloid DC, TLR were dispensable for TNF-alpha and IL-12 induction by bacterial RNA. Even non-immune stroma cells were able to mount a NF-kappaB response upon triggering with bacterial RNA. Retinoic-acid inducible gene I and melanoma-differentiation-associated gene 5 could be ruled out to be responsible for this reactivity. Although the inflammasome adaptor protein, apoptosis-associated speck-like protein, and a functional type-I IFN receptor were necessary for IL-1beta secretion in myeloid DC, these proteins were dispensable for TNF-alpha and IL-12 induction by cytosolic bacterial RNA. Our results show that besides of activation of TLR7 and inflammasomes, bacterial RNA activates additional cytosolic receptors similarly as has been reported for recognition of bacterial DNA.