Seventy-one cases of invasive squamous cell carcinoma (ISCC) of the vulva were compared with 18 cases of vulvar intraepithelial neoplasia (VIN) and 21 cases of lichen sclerosus. Ploidy was studied by image analysis, HPV-DNA by PCR, and p53 and pRb by immunohistochemistry. Univariate and multivariate statistical analyses were performed. The mean age of the patients with ISCC was 70.6 years; only 8.5% were < 60 (range, 43-89) years. For the 43 patients with follow-up, FIGO surgical stages were I in 16.2%, II in 48.8%, III in 27.9%, and IV in 6.9%. The 5-year survival was 90% for the patients with curative surgery (vulvectomy and lymphadenectomy) and 32% for those with tumors in stages III to IV. Previous history of nonneoplastic epithelial alterations was recorded in 54%. Vascular invasion was detected in 4.3% and perineural invasion in 21.4%. Inguinal lymph node metastases were present in 34.9% of the cases. Fifty-one (72%) ISCCs were aneuploid, HPV-DNA-16 was detected in 7 (12.3%) cases, overexpression of p53 was found in 40 (56%), and pRb expression was negative in 15 (21.4%). Fifteen cases (80%) of VIN were aneuploid, 5 (27.7%) contained HPV-DNA, 11 (61%) were positive for p53, and all immunoreacted for pRb. All lichen sclerosus cases were diploid, did not contain HPV-DNA, failed to stain for p53, and were positive for pRb. Our study confirmed the prognostic value of conventional pathological features: stage, lymph node metastasis, histological grade, and vascular and perineural invasion; all were statistically significant for survival in the univariate analysis. Also, ploidy was significant in patients with stages I and II tumors. The only significant variable in the multivariate analysis was stage. p53 overexpression appears as a late event in vulvar carcinogenesis, but it may occur before tumor invasion. Lack of pRb expression can occur in vulvar neoplasia, but it does not seem to play any role in the initiation or prognosis of vulvar ISCC.