Human papillomavirus (HPV) status in cervical smears from cervical intraepithelial neoplasia (CIN) 2/3 diagnosed in 36 of 892 women with a history of normal cytology and colposcopy (incident CIN 2/3) was compared with CIN 2/3 in 40 patients with a history of abnormal cytology (prevalent CIN 2/3). In all patients cervical smears for HPV testing and cytology and two cervigrams were taken. The scrapes were collected in hybrid capture assay solution and analyzed with the hybrid capture and general primer/type-specific primer polymerase chain reaction system (GP/TS-PCR) after DNA extraction. Patients with incident and prevalent CIN 2/3 were similar with respect to age. By GP/TS-PCR carried out under suboptimal conditions due to DNA extraction, HPV DNA was detected in 69.4% (25 of 36) of smears from incident CIN 2/3 compared to 95% (38 of 40) in prevalent CIN 2/3 (P = 0.003). Using hybrid capture, smears of incident CIN 2/3 were HPV positive in 50% (18 of 36) compared to 80% (32 of 40) in prevalent CIN 2/3 (P = 0.006). High-risk HPVs were significantly less common in smears from incident CIN 2/3 compared with prevalent CIN 2/3: 36.1% vs 72.5% by GP/TS-PCR (P = 0.001) and 47.2% vs 80% by hybrid capture assay (P = 0.003), respectively. Virus load in HPV-positive smears of prevalent CIN 2/3 was significantly higher than of incident CIN 2/3 using semiquantitative PCR (P = 0.0005). Thus, high-risk HPV types were detected less frequently and in lower concentration in smears from incident CIN 2/3 than in smears from prevalent CIN 2/3.