Protein expression of human toll-like receptors (TLR) 1-10 was measured in cell lines and solid tumors of head and neck squamous cell carcinoma (HNSCC). All HNSCC cell lines and 80% of solid tumors were found to express TLR3 as a predominantly intracellular protein, while no other TLR proteins were expressed. TLR3 has previously been shown to contribute to the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor which promotes several types of human cancers. Significantly, NF-kappaB expression was strongest in protein extracts from carcinoma tissue in which TLR3 was overexpressed. Inhibition of TLR3 expression in permanent HNSCC cell lines resulted in decreased expression of the oncoprotein c-Myc resulting in decreased cell proliferation. Correspondingly, overexpression of human TLR3 in mouse fibroblasts resulted in an upregulation of c-Myc and increased sensitivity for PolyI:C-induced cell proliferation. Our data suggest that TLR3 contributes to the malignant phenotype leading to invasive carcinoma in HNSCC.