Background: Treatment with a blocking αPD-1 antibody recently showed clinical efficacy for various tumors types. In this study, we characterized the expression profile of PD-1/PD-L1 and the potential of PD-1 blockade in human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC). Methods: Lymphocytes from peripheral blood, draining lymph nodes and the tumor were phenotyped for PD-1 expression, and their proliferative activity was assessed in the presence of blocking αPD-1 treatment. Primary tumor expression of PD-L1 was also analyzed using immunohistochemistry. Results: Lymphocyte PD-1 expression was abundant with highest expression in the tumor and in vitro mixed lymphocyte reaction demonstrated that PD-1 blockade could induce T cell proliferation. Furthermore, tumor cells were found to have three distinct patterns of PD-L1 expression with over 82% of the specimens demonstrating strong PD-L1 positivity. Conclusions: Our data strongly supports the use of αPD-1 blockade in HPV-negative HNSCC patients that are refractory to standard treatments. Head Neck, 2014.