Human papillomavirus (HPV) E6/E7 mRNA has been proposed as a more specific marker for cervical dysplasia and cancer than HPV DNA. This study evaluated the RNA specificity of nucleic acid sequence-based amplification (NASBA)-based HPV detection using HPV DNA plasmids (HPV type 16 [HPV16], HPV18, HPV31, HPV33, and HPV45) and nucleic acid extracts of several cell lines, which were systematically subjected to enzymatic treatments with DNase and RNase. HPV plasmid dilutions (10(6) to 10(0) copies/microl) and nucleic acid extracts (total DNA, RNA-free DNA, total RNA, and DNA-free RNA) of unfixed and fixed (PreServCyt and SurePath) HaCaT, HeLa, and CaSki cells were tested with the NucliSENS EasyQ HPV test. The RNA-free DNA extracts of HeLa and CaSki cells could be amplified by HPV18 and -16 NASBA, respectively. Fixation of the cells did not influence NASBA. All HPV plasmids could be detected with NASBA. Based on the plasmid dilution series, a lower detection limit of 5 x 10(3) HPV DNA copies could be determined. Our study identified viral double-stranded DNA as a possible target for NASBA-based HPV detection. The differences in diagnostic accuracy between the NASBA-based tests and conventional HPV DNA detection assays seem to be attributable not to the more specific amplification of viral mRNA but to the limited type range and the lower analytical sensitivity for HPV DNA.