Vulvar intraepithelial neoplasias (VINs) are potentially premalignant lesions of the squamous mucosa. The immunohistochemical distribution of the catalytic protein subunit of telomerase (hTERT) and the patterns of X chromosome inactivation were investigated as markers of neoplasia in samples from a patient with multifocal and diffuse VIN. hTERT nuclear staining in VIN correlated with squamous maturation and the degree of nuclear atypia. Normal mucosa revealed faint nuclear staining of parabasal cells and lower intermediate layer squamous cells. Monoclonal composition was demonstrated in 0 of 3 samples of VIN1, 2 of 3 samples of VIN2, and 13 of 13 samples of VIN3. The patterns of X chromosome inactivation indicated intramucosal extension and multifocal origin of individual lesions. Five samples of histologically normal vulvar squamous epithelium revealed a random pattern of X chromosome inactivation, consistent with polyclonal composition. All 19 samples from 9 lesions contained human papillomavirus (HPV)-16 sequences. Neither mutations in the p53 tumor suppressor gene or K-ras oncogenes nor loss of heterozygosity at 7 chromosomal loci were detected in any of the 19 samples of VIN. These results demonstrate that HPV-associated VIN may result from multifocal and diffuse 2-dimensional intraepithelial expansion of an immortalized monoclonal cell population.