Monitoring human papillomavirus (HPV) E6/E7 mRNA expression may provide an accurate and informative diagnostic approach for detection of oncogene activity related to the development of severe dysplasia or cervical carcinoma. A multiplex nucleic acid sequence based amplification (NASBA) assay, utilizing molecular beacon probes for real-time detection was developed for the identification of E6/E7 mRNA from HPV types 16, 18, 31, 33 and 45. The assay is called PreTect HPV-Proofer and this report describes the development and the analytical performance of the assay. The reproducibility of PreTect HPV-Proofer with regard to a positive result was found to be between 96 and 100%, depending on HPV type. The melting temperature for the different molecular beacons was in the range of 48-55 degrees C, indicating conformational stability, i.e. the molecular beacons will not get activated by the 41 degrees C annealing temperature, but will be activated by the annealing to the target itself. The limit of detection for HPV 16 was ten SiHa or CaSki cells and for HPV 18 one HeLa cell. No cross reactivity was observed with E6/E7 mRNA from the other tested HPV types. mRNA from cervical cells was also successfully amplified after more than one year of storage. In conclusion, the PreTect HPV-Proofer assay, individually identifying E6/E7 mRNA expression from five carcinogenic HPV types, is a reproducible assay that may serve as a valuable tool in monitoring HPV infections producing proteins with a transforming potential.