Archival paraffin-embedded tumor specimens offer a wealth of information for both cancer research and for routine clinical applications. However, the use of formalin-fixed, paraffin-embedded specimens for quantitative real-time PCR is not yet a standard diagnostic method in many laboratories, in particular for the quantification of human papillomavirus (HPV). Particularly high-risk HPV types are involved in almost 100% of the carcinogenesis of cervical cancer. We compared the diagnostic applicability and sensitivity of real-time PCR to that of chromogenic tyramide-signal-amplified in situ hybridization and conventional PCR for the detection of HPV from archival tissue in 164 cases of carcinoma in situ and cervical cancer. Furthermore, we examined whether the viral load of HPV is of prognostic relevance. Our findings indicate that patients in tumor stage I with a lower viral load of HPV type 16 (HPV16; up to 1,000 copies/ng of DNA) had a significantly better survival than HPV 16-negative patients (P = 0.037). We observed a greater sensitivity of both real-time PCR and conventional PCR for the detection of HPV16 and -18 compared to signal amplified in situ hybridization. We found a considerable concordance between HPV16 (kappa = 0.661) and HPV18 (kappa = 0.781) status as measured by real-time PCR and conventional PCR, indicating similar sensitivities. We recognized an inhibitory effect of formalin fixation and paraffin embedding on the evaluation of real-time PCR quantification.