BACKGROUND.: Few human papillomavirus (HPV)(+) head and neck squamous cell carcinoma (HNSCC) cell lines exist. We established University of Michigan-squamous cell carcinoma-104 (UM-SCC-104), a new HPV(+) HNSCC cell line from a recurrent oral cavity tumor, and characterized it for the presence of cancer stem cells (CSCs). METHODS.: Tumor cells were tested for biomarker expression by immunohistology, and the presence of HPV was assessed by several methods. RESULTS.: UM-SCC-104 has a unique genotype, contains HPV-16, and expresses E6/E7. Inoculation of aldehyde dehydrogenase (ALDH)(+) and ALDH(−) cells in an immunocompromised mouse resulted in tumor growth from the ALDH(+) cells after 6 weeks that recapitulated the histology of the primary, whereas ALDH(−) cells did not produce tumors. CONCLUSION.: UM-SCC-104, a new HPV-16, CSC-containing HNSCC cell line will aid in studying recurrent HPV(+) tumors. The aggressive nature of this tumor is consistent with high uniform expression of epidermal growth factor receptor (EGFR) and a functionally significant proportion of ALDH(+) CSCs. © 2011 Wiley Periodicals, Inc. Head Neck, 2011