Technical Abstract: In the spring of 2009, a novel H1N1 influenza A virus began to spread among humans worldwide. The genomic features of the new pandemic H1N1 were immediately identified: it contained gene segments with ancestors in North American and Eurasian swine influenza virus (SIV) lineages providing the virus a unique genetic constellation as well as genomic markers. Although the new H1N1 has been demonstrated recently to be circulating among pigs, human infection was not connected to pig exposure. However, if the virus begins to widely circulate in pigs, the human population may be at increased risk because of the potential reservoir represented by the swine population. In addition, viral reassortment may be possible in the swine host, which has been previously recognized as a mixing vessel for avian and human influenza viruses. In order to investigate the potential outbreak of the new pandemic virus in pigs, a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of the 2009 pandemic H1N1 RNA in clinical specimens was developed. To evaluate the applicability of the test as a diagnostic tool in the screening of field specimens from swine, we retrospectively analyzed field isolates of swine, equine and avian influenza viruses collected during diagnostic investigations as well as 116 samples collected during an experimental in vivo infection with two novel H1N1 isolates, A/California/04/2009 (H1N1)v and A/Mexico/4108/2009 (H1N1)v. Our unique and highly sensitive test represents a rapid and useful approach for the screening and quantitation of the novel 2009 H1N1 RNA in clinical specimens.