Bacteria and viruses may be transmitted to laboratory rodents by contaminated biological materials such as transplantable tumours, cell lines, sera or other biological materials. Biological materials are currently being screened using the mouse or rat antibody production (MAP/RAP) test (serological testing). We decided to test and validate an alternative assay using polymerase chain reaction (PCR/realtime PCR) technology to detect viral contamination directly in biological material. The aim of this study therefore is the validation of our new PCR assays and the comparison of PCR and the MAP test. For 8/14 viruses, conventional PCR was more sensitive and more specific than the MAP test in detecting murine viruses. For 12/14 viruses, the realtime PCR was more sensitive than the MAP test. In 2/14 cases, all three detection methods had the same sensitivity. Furthermore, PCR screening clearly conforms to the principles of the 3Rs as a replacement technique because it eliminates the need for using animals to screen for murine viruses in biological material.