One of the most interesting aspects of real-time PCR based on the detection of fluorophoric labeled oligonucleotides is the possibility of being able to detect conveniently multiple targets in the same PCR reaction. Recently, Roche Diagnostics launched a real-time PCR platform, the LightCycler480 (LC480), which should be well suited for multiplex real-time PCR analysis. In this paper the performance of the LC480 and accompanying software for the detection of five different targets was analyzed. Target DNAs mixed at equimolar concentrations were detected reproducibly and quantitatively. In addition, mixing different concentrations of the five targets demonstrated that the LC480 is capable of providing quantitative results for a mixture of DNA sequences without losing sensitivity. When applied to the practice of molecular diagnosis of four respiratory viral infections the multiplex assay showed almost complete concordance with corresponding single-target PCRs. The application of multiplex PCR for the detection of multiple pathogens within the same sample will provide a major contribution to the efficiency, logistics and cost-effectiveness of molecular diagnostics.