The protein products of the two tumor suppressor genes located on 9p and 13p, p16INK4 and Rb, respectively, play an important role in regulation of the cell cycle and are implicated in tumorigenesis. We examined 49 cases of benign vulvar lesions, vulvar intraepithelial neoplasia (VIN), and squamous cell carcinoma with immunohistochemical staining to determine expression of p16INK4 and Rb. All and 86% of benign lesions expressed Rb and p16INK4, respectively; 40% each of VIN I and VIN III expressed p16INK4 and Rb, respectively; and 37 and 68% of squamous cell carcinomas expressed p16INK4 and Rb, respectively. The combination of the lack of p16INK4 and/or Rb expression increased from benign lesions (14.3%), through VIN I (60%) and VIN III (60%), to invasive squamous cell carcinoma (72%), thus supporting the postulation that alterations in p16INK4 or Rb could be significant events in progression of disease. The loss of Rb expression also increased from stage I carcinoma (16.7%) through stage II (26.7%) and III (44.4%), to IV (50%), suggesting that Rb may play an important role in tumor progression. A larger study on VIN lesions and genetic coding is suggested to further investigate the role of p16INK4, Rb, and other factors in tumorigenesis and progression of vulvar cancers.