Previous studies of vulvar carcinomas have shown two distinct subsets with respect to several clinicopathologic features. In younger women, the tumors are frequently human papillomavirus (HPV) positive, are usually of basaloid or warty histology, and are associated with vulvar intraepithelial neoplasia. In older women, the tumors are usually HPV negative, are typical keratinizing squamous carcinomas, and are associated with squamous hyperplasia--a lesion that has been purported to serve as a precursor to HPV-negative invasive carcinoma. In squamous carcinomas of the cervix, p53 inactivation (through gene mutation or interaction with the HPV E6 oncoprotein) occurs in most cases. Comparatively few studies have assessed p53 mutation and HPV status in vulvar carcinomas, and none has used molecular markers to evaluate squamous hyperplasias as direct precursors of HPV-negative invasive cancers. Of 18 invasive squamous carcinomas analyzed, seven (39%) were found to be HPV positive. Four p53 gene mutations were identified--all in HPV-negative tumors. DNA was subsequently prepared from microdissected archival tissues from all four specimens showing p53 gene mutations. DNA was separately isolated from normal squamous epithelium, invasive squamous carcinoma, and associated squamous hyperplasia. In each specimen, the p53 mutation was confirmed in the invasive tumor and absent in both normal and hyperplastic epithelium. To further investigate squamous hyperplasia as a potential precursor of HPV-negative invasive carcinoma, the authors determined the clonality of hyperplastic lesions adjacent to invasive carcinomas with p53 mutation. Clonality analyses were performed using a polymerase chain reaction (PCR)-based assay for X chromosome inactivation. Although all three informative carcinomas tested were monoclonal, corresponding normal epithelia and hyperplastic lesions were polyclonal. These findings underscore the heterogeneity of vulvar cancers with respect to loss of wild type p53 function either by interaction with the HPV E6 oncoprotein or somatic mutation of p53, and suggest that squamous hyperplasias do not serve as direct precursors of HPV-negative squamous carcinomas.