Human papillomavirus (HPV) infection is associated with high-grade vulvar intraepithelial neoplasia (VIN-3). The prevalence of anogenital HPV infection in women with previously treated VIN-3 has not been documented yet. This cross-sectional study compared high-risk HPV DNA detection rates in women with past (n = 30) and current (n = 22) VIN-3 to those without current or past VIN (n = 86). HPV DNA was detected in vulvar and cervical samples with Hybrid Capture 2 (HC-2). Smoking was associated in multivariate analysis with current VIN-3 (odds ratio (OR) 8.3, 95% confidence interval (CI) 2.0–8.2) and any VIN-3 history (OR 6.5, 95% CI 2.5–16.5). High-risk HPV DNA was found on the vulva of 64%, 33%, and 20% of women with current VIN-3, past VIN-3, and without previous or current VIN, respectively. After controlling for age and smoking, high-risk HPV vulvar infection was associated with cervical high-risk HPV infection (OR 8.6, 95% CI 2.8–26.5; P = 0.001). After controlling for age, HPV infection was more often multifocal in women with current VIN-3 compared to women with previous but no current VIN-3 lesion (OR 17.6, 95% CI 1.4–227.2). Multifocal vulvar HPV infection was detected in women with previous or active VIN-3. Longitudinal studies are required to determine if the multifocality of HPV infection on the vulva could explain the high recurrence rate of VIN-3. J. Med. Virol. 78:814–819, 2006. © 2006 Wiley-Liss, Inc.