This study was designed to identify the prevalence of human papillomavirus 16 (HPV 16) in oral exfoliated cells from 26 patients with oral cancer and matched healthy volunteers with the use of polymerase chain reaction. In addition, the value of a silver staining technique for nucleolar organizer regions (AgNORs) was also investigated. HPV 16 was detected in 30.8% of the cancer lesions, 26.9% of the unaffected sites, and 15.4% of samples from normal mucosa. AgNOR counts on the same cases were analyzed. Although AgNOR counts are useful in distinguishing between normal and malignant oral exfoliated cells, they provided no additional prognostic information for oral cancer. However, when AgNOR counts were compared with HPV 16-positive and HPV 16-negative counts in cancer lesions, AgNOR counts were higher in HPV-positive lesions. These findings suggest that HPV 16 may play a role in tumor cell proliferation, but it is unlikely to play a significant role alone in the cause of oral cancer. Therefore evidence of HPV 16 infection in oral malignant neoplasms should be cautiously interpreted.