Pulmonary airways are vulnerable to infection because of exposure to Ag during respiration. The innate, antiviral response must be activated rapidly after pathogen recognition, and alveolar macrophages (AMphi) play a role in this response. TLR3 and protein kinase R (PKR) recognize dsRNA, a replication intermediate of RNA viruses, and initiate transcription of IFN-alphabeta. In this study, synthetic dsRNA poly(I:C) was used to investigate innate responses of porcine AMphi compared with responses of peritoneal macrophages (PMphi). Poly(I:C) triggered IFN-alphabeta in AMphi and PMphi, but levels in AMphi were higher. In contrast, mRNA levels of IFN-stimulated genes, Mx and PKR, were greater in PMphi than AMphi. Low levels of Mx and PKR transcription in AMphi were not due to deficient type I IFN receptor signaling, as exogenous IFN-alpha induced nuclear translocation of phosphorylated STAT1. To investigate the differential mechanism by which IFN-alphabeta transcription is activated in AMphi and PMphi, 2-aminopurine (2-AP) was used to block dsRNA-mediated activation of PKR. IFN-alphabeta, Mx, and PKR mRNA levels in AMphi after poly(I:C) treatment were unaffected by 2-AP; conversely, transcription of IFN-alphabeta, Mx, or PKR remained at baseline levels in PMphi. Phosphorylated PKR was detected in PMphi, but not AMphi, after poly(I:C) treatment. In addition to IFN-alphabeta gene induction, mRNA levels of TNF-alpha and RANTES were higher in AMphi than PMphi after poly(I:C) stimulation. In summary, differential dsRNA-induced cytokine expression patterns between AMphi and PMphi provide evidence that dsRNA recognition and subsequent signaling is likely mediated via TLR3 in AMphi and PKR in PMphi.