BACKGROUND Recent evidence has accumulated suggesting that human papillomavirus (HPV) plays a role in the development of head and neck squamous cell carcinoma (HNSCC). HPV16 is the most common of the HPV subtypes associated with oral and laryngeal malignancies. This study estimated the prevalence of HPV16 DNA in Puerto Rican patients with HNSCC. METHODS DNA was extracted from frozen tissue of 118 HNSCCs. Genomic DNA was screened for the presence of HPV16 DNA with E6-specific and E7-specific primers. RESULTS HPV16 was detected in tumor tissue of 52 patients (44%) with HNSCC. The oropharynx had a slightly higher incidence of HPV16 DNA. Fifteen of 66 patients with HPV16-negative HNSCC later had recurrences. Positivity for HPV16 was independent of the tumor grade, tumor stage, nodal status, and tobacco or alcohol use. The 3-year survival rate was higher in HPV16-positive patients than in HPV16-negative patients (36% vs 21%). CONCLUSIONS Our findings suggest that HPV16 may play a role in the etiology of a subgroup of HNSCC in Puerto Ricans. Overall survival times of the HPV16-positive patients were not significantly different from those of HPV16-negative patients. Increasing our understanding of the role of HPV16 in the etiology of HNSCC might facilitate the development of new treatment modalities for this subgroup of HNSCC.