Recent evidence suggests that loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene plays a role in colorectal tumorigenesis and other cancers. However, little is known as to whether the APC gene contributes to the pathogenesis of oral squamous cell carcinoma. To assess involvement of both the APC gene and the human papillomavirus (HPV) in the development of oral pre-malignant and malignant lesions, we analysed DNA from 14 paired oral normal and pre-malignant or malignant paraffin-embedded biopsy specimens, and DNA from cultured normal and HPV 16-immortalised oral epithelial cells for the presence of LOH of APC and for HPV infection, using PCR based techniques. LOH of APC occurred in 80% of cases of oral epithelial dysplasia, 67% of carcinoma in situ, 50% of invasive squamous cell carcinoma cases, and in the HPV 16-immortalised oral epithelial cells. HPV was detected in half of the biopsy specimens, with HPV 16 as the dominant type. More than half of the carcinoma cases were found to contain both LOH of APC and HPV infection. These results suggest that LOH of APC is an early event during oral tumorigenesis. Our findings also suggest a strong correlation between HPV infection, particularly HPV 16, and LOH of the APC gene in oral squamous cell carcinomas.