A role for cutaneous human papillomaviruses (HPV) has been proposed in the development of skin cancer. Well-designed epidemiologic studies to demonstrate an association between HPV infection and skin cancer are extremely rare. To identify HPV infection as a potential risk factor, we investigated the association between the presence of HPV DNA in eyebrow hairs and a history of cutaneous squamous cell carcinoma. A case-control study was designed consisting of 155 immunocompetent individuals with a history of squamous cell carcinoma and 371 controls without skin cancer. DNA extracted from plucked eyebrow hairs collected from the study population was analyzed with a cutaneous HPV subgroup polymerase chain reaction and newly designed HPV type specific polymerase chain reactions for HPV 2, 5, 8, 15, 16, 20, 24, and 38. HPV DNA was detected in 63.1% of the total study population. The presence of HPV DNA was associated with age (p=0.0002) and male sex (p=0.02), but not with sun exposure, skin type, and smoking. After adjustment for age and sex, the presence of HPV DNA in eyebrow hairs was associated with a history of squamous cell carcinoma (odds ratio 1.7, 95% confidence interval 1.1; 2.7). HPV type specific analysis revealed that no HPV type stood out. The high-risk mucosal type HPV 16 and the skin wart type HPV 2 were rarely found in this study (<0.2%). The positive association found between the presence of HPV DNA in eyebrow hairs and a history of squamous cell carcinoma warrants further research into the role that HPV infection plays in the development of cutaneous squamous cell carcinoma.