Women with abnormal smears have an increased risk of developing cervical cancer. During the 8 years following conservative treatment of cervical intraepithelial neoplasia (CIN), their risk of invasive cervical cancer is about 5 times greater than that of the general population. Human papillomavirus (HPV) has been associated with the natural history of both CIN and cervical cancer. To date, there have been no published reports on the predictive value of HPV testing in the treatment outcome of CIN. A prospective, multi-center, cohort study was conducted on women in the Northwest of England who were attending for treatment of CIN. They were asked to complete a questionnaire, which included a detailed smoking history. Pre- and post-treatment HPV testing was performed on cervical biopsies and cervical swab, being taken with the first follow-up smear at 6 months. A nested case-control analysis was performed, cases being defined as women who developed CIN within the 2 years of treatment and controls being sampled from those who did not experience treatment failure within 2 years. Multiple conditional logistic regression is used to study the factors associated with treatment failure of CIN. The cohort included 958 women of whom 77 (8%) experienced treatment failure (cases). Two controls were matched to each case (154). Smoking status was significantly associated with CIN treatment failure(p= 0.0013). Current smokers had a 3-fold increased risk of treatment failure of CIN as compared to non-smokers (95% CI 1.65 to 5.91). Five hundred twenty-five women underwent HPV sampling following treatment, of whom 47 (8.9%) developed further CIN. Post-treatment positive HPV testing was found to be strongly associated with treatment failure of CIN (OR 23.3; 95% CI 3.15-172.1). In 11/45 cases with negative smear at first follow-up, the HPV test was positive. The combination of both HPV and cytology in the first follow-up visit predicted treatment failure in 72% of the cases. Cigarette smoking is a factor, which, independently of HPV infection, influences the treatment outcome of CIN. Smokers and those who are HPV positive during follow-up appear to require longer, more intensive follow-up. HPV testing requires careful consideration as part of routine follow-up protocol following treatment of CIN.