Background Human papillomavirus type 16 (HPV16) seropositivity and alcohol and tobacco use have been associated with risk of head and neck squamous cell carcinoma (HNSCC). However, it is less clear whether HPV16 influences HNSCC risk associated with alcohol and tobacco use. Methods Incident cases of HNSCC diagnosed between December 1999 and December 2003 were identified from nine medical facilities in Greater Boston, MA. Control subjects were frequency matched to case subjects on age, sex, and town of residence. A total of 485 case subjects and 549 control subjects reported information on lifetime smoking and alcohol consumption and provided sera, which was used to determine presence of HPV16 antibodies. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) of HNSCC risk by alcohol consumption (drinks per week: <3, 3 to <8, 8 to <25, ≥25) and smoking (pack-years: none, >0 to <20, 20 to <45, ≥45), adjusting for age, sex, race, education, and HPV16 serology. Polytomous logistic regression was used to estimate odds ratios and 95% confidence intervals for the association of HPV16 serology, alcohol consumption, and tobacco use in site-specific analyses. All statistical tests were two-sided. Results The strongest risk factors by tumor site were smoking for laryngeal cancer, alcohol for cancer of the oral cavity, and HPV16 for pharyngeal cancer. For pharyngeal cancer, risk increased with increasing alcohol consumption (OR≥25 versus <3 drinks per week = 5.1, 95% CI = 2.4 to 11.0) and smoking (OR≥45 pack-years versus never smoker = 6.9, 95% CI = 3.1 to 15.1) among HPV16-seronegative subjects but not among HPV16-seropositive subjects (Pinteraction, HPV16 serology and alcohol = .002; Pinteraction, HPV16 serology and smoking = .007). Among light drinkers or never smokers, HPV16 seropositivity was associated with a 30-fold increased risk of pharyngeal cancer. Conclusions Alcohol or tobacco use does not further increase risk of HPV16-associated pharyngeal cancer. HNSCC risk associated with smoking, alcohol, and HPV16 differs by tumor site.