A new human coronavirus, subsequently named Middle East respiratory syndrome coronavirus (MERS-CoV), was first reported in Saudi Arabia in September, 2012. In response, we developed two real-time reverse transcription-polymerase chain reaction (rRT-PCR) assays targeting the MERS-CoV nucleocapisd (N) gene and evaluated these assays as a panel with a previously published assay targeting the region upstream of the MERS-CoV envelope gene (upE) for detection and confirmation of MERS-CoV infection. All assays detected 10 or fewer copies/reaction of quantified RNA transcripts with a linear dynamic range of 8 log units and 1.3 x 10-3 TCID50/mL of cultured MERS-CoV per reaction. All assays gave comparable performance with respiratory specimens, sera and stool spiked with cultured virus. No false positive amplifications were obtained with other human coronaviruses or common respiratory viral pathogens or with 336 diverse clinical specimens from non-MERS-CoV cases; specimens from two confirmed MERS-CoV cases were positive by all assay signatures. In June, 2012, the U.S. Food and Drug Administration authorized emergency use of the rRT-PCR assay panel as an in vitro diagnostic test for MERS-CoV. A kit consisting of the three assay signatures and positive control were assembled and distributed to public health laboratories in the U.S. and internationally to support MERS-CoV surveillance and public health response.