A quantitative two-step multiplex real-time reverse transcriptase (RT-) PCR assay for the simultaneous detection of genogroup I (GI) and genogroup II (GII) noroviruses (NoVs) is described below. A murine norovirus 1 (MNV-1) real-time PCR detection assay described recently was integrated successfully into the multiplex assay, making it possible to detect GI and GII NoVs and MNV-1 in one reaction tube with MNV-1 plasmid DNA as real-time PCR internal amplification control (IAC). The results showed a nearly complete concordance between the multiplex assay and the corresponding single-target PCRs. Analysis of competition between the individual reactions within the multiplex real-time PCR assay showed that GI and GII NoV plasmid DNAs mixed at equimolar concentrations were detected reproducibly and quantitatively, while a 4 log excess between GI and GII plasmid DNAs hindered amplification of the target with the lowest concentration. High concentrations of the real-time PCR IAC (MNV-1 plasmid DNA) also interfered with the possibility of the developed multiplex real-time RT-PCR assay to detect quantitatively and simultaneously the presence of GI and GII NoVs within one sample. The specificity of the multiplex assay was evaluated by testing a NoV RNA reference panel containing nine GI, eight GII, and one GIV in vitro synthesized RNA fragment, plus 16 clinical samples found positive for GI and GII NoVs previously. In addition, a collection of bovine NoVs and other (non-NoV) enteric viruses were found to be negative, and no cross-amplification between genogroups was observed.