Using a case control approach, we performed a two-way comparison study between GP5+/6+-PCR and HPV SPF(10)-Line Blot 25 (SPF(10)) assays for detection of 14 types of high-risk human papillomavirus (hrHPV) in samples from women with normal cytology results who had or developed grade 3 cervical intraepithelial neoplasia (CIN 3). Samples were pooled from two cohorts, i.e., women participating in population-based screening and women attending a gynecological outpatient clinic. Cases (n = 45) were women with histologically confirmed CIN 3 diagnosed within a median follow-up time of 2.7 (range, 0.2 to 7.9) years. Control samples were from women (n = 264) who had developed CIN 1 lesions at maximum (median follow-up at 5.8 [range, 0 to 10] years). Identical numbers of cases tested positive for 1 or more of the 14 hrHPV types by both systems (40/45; McNemar; P = 1.0). Conversely, SPF(10) scored significantly more controls as hrHPV positive than did GP5+/6+-PCR (95/264 versus 29/264; McNemar; P < 0.001). Consequently, women with normal cytology results and an hrHPV GP5+/6+-PCR-positive test exhibited a risk of CIN 3 that was 4.5 times higher (odds ratio [OR], 65; 95% confidence interval [95%CI], 24 to 178) than that seen for women with an hrHPV-positive SPF(10) test (OR, 14; 95%CI, 5 to 38)). Similar results were obtained after analysis of both cohorts separately. Discrepancy analysis by viral load assessment for the most common discordant hrHPV types (HPV16, -18, and -52) showed that samples which were SPF(10) positive only for these types had viral loads significantly lower than those for samples that were positive by both assays (analysis of variance; P < or = 0.006). Our data indicate that GP5+/6+-PCR has a better clinical performance than SPF(10) for women who are diagnosed with CIN 3 after prior normal cytology results. The extra positivity scored by SPF(10) mainly involved infections characterized by low viral loads that do not result in CIN 3.