The most common neoplasm arising in the upper aerodigestive tract is head and neck squamous cell carcinoma (HNSCC). Tumor growth, invasion and systemic dissemination is a multistep process of dysregulated cellular signaling pathways and an altered cell-cell and cell-matrix interaction. Aberrant Wnt/β-catenin signaling is linked to tumor development and dissemination in several tumor entities. β-catenin is a multifunctional protein within the canonical Wnt pathway, which is an important factor for reducing cell-cell adhesion in malignant tissue and for triggering cell cycle progression and unscheduled proliferation. Another pivotal factor in carcinogenesis is the tyrosine kinase receptor c-kit, which in the case of dysregulated expression is associated with neoplastic transformation in epithelial tissue. This study evaluates the expression pattern of secreted and nuclear β-catenin and c-kit in p16-positive and HPV-negative squamous cell carcinomas (SCC) and the vulnerability of therapy with the tyrosine kinase inhibitor imatinib as a potential targeted treatment modality compared to platinum-based chemotherapeutic drugs. The different squamous tumor cell lines were incubated with increasing concentrations of carboplatin (3 or 7.5 µmol/ml) and imatinib (18 or 30 µmol/ml). ELISA and immunohistochemical methods were carried out after 48, 72, 120, 192 and 240 h. We detected a reliable trend towards significantly decreased cytosolic and nuclear β-catenin and c-kit expression levels in p16-positive SCC and non-HPV HNSCC cells induced by imatinib exposure for an extended incubation period, whereas platinum-based agents had no or, at best, a slight influence. Virus-transformed squamous cell carcinoma (CERV196) cells were characterized by a reduced susceptibility to an imatinib-altered β-catenin expression. Further studies are planned to investigate this observance in HPV-positive HNSCC in vitro. The implementation of a selective molecular therapy in established chemotherapeutic regimes may enhance the efficacy of platinum-based chemotherapy without increased toxicity and could thus improve the clinical outcome in HNSCC, irrespective of the HPV status.