Plasmacytoid dendritic cells (pDCs) recognize RNA virus infection via TLRs and consequently produce vast amounts of type I IFN. Because nucleic acid-sensing TLRs reside in the intracellular membrane compartment, it is presumable that pDCs do not require cytoplasmic viral replication to recognize the infection. By checking Newcastle disease virus (NDV) RNA abundance in GFP+ and GFP- pDCs from Ifna6gfp mice, we found that NDV replication was not detected in IFN-producing pDCs. GFP- pDC was induced in response to replication-incompetent NDV. In contrast, the replication-incompetent NDV failed to induce IFN-producing pDCs in type I IFNR-deficient mice. The lack of IFNR signaling led to the replication of NDV and the subsequent RIG-I-like helicase-dependent IFN-α production in pDCs. These results showed that detection of viruses via TLRs together with a type I IFN feedback system circumvents the requirement for viral replication-dependent recognition in pDCs. Copyright © 2009 by The American Association of Immunologists, Inc.