Osteopontin (OPN) is a secreted glycoprotein implicated to function in cancer development and metastasis. Although elevated expression of OPN are observed in cancer cells of various types, in some cases, only the cells in the stromal region surrounding the tumor express OPN, suggesting distinct functional roles for this protein derived from host cells and from cancer cells. To provide a model for addressing the functions and mechanisms of host-derived OPN in cancer progression and metastasis, a cutaneous squamous cell carcinoma cell line (ONSC) that lacks the OPN gene, Spp1, was established. This line of cells was derived from a squamous cell carcinoma that developed in a female, OPN-null mouse subjected to two-stage skin carcinogenesis. Morphologically, ONSC cells resemble epithelial cells, and they express the epithelial markers, K1, K14, and p63, as confirmed by immunohistochemical analyses. Genomic analyses indicate the presence of mutated H-Ras and p53 genes. ONSC cells form colonies in soft agar and, subcutaneously injected into athymic nude mice, develop into squamous cell carcinomas that metastasize to the lungs. Lacking OPN expression, these squamous cell carcinoma cells provide a model to address the function of host OPN in the context of cancer progression and metastasis. © 2009 The Society for In Vitro Biology.