Background: Activation of RAS/MAPK/PI3K pathway is implicated in resistance to EGFR-targeted therapy in cancer. The prevalence of H-RAS and PIK3CA mutations has not been studied extensively in Caucasian populations. We sought to determine the impact of H-ras and PIK3CA gene mutations on resistance to cetuximab in head and neck cancer. Methods: We performed mutation analysis for H-RAS and PIK3CA genes in a cohort of 105 untreated HNSCC patients. To investigate whether PI3K/HRAS mutations in head and neck cancer can lead to resistance to EGFR inhibitors, we treated HNSCC cell lines Cal-33 (PIK3CA mutation), HSC-2 (PIK3CA mutation), BB49 (HRAS mutation) and A431 cells with increasing concentrations of cetuximab alone, PI3K inhibitor (LY294002) alone or both and performed MTT cell viability assay 72 hours later. We subsequently treated tumor xenografts derived from the aforementioned cell lines with cetuximab or LY294002 or both and measured tumor volume 4 weeks later. Results: We found that 30 of 105 (29%) HNSCC contained H-ras molecular alterations and 4 of 105 (4%) PIK3CA mutations. Eight of 30 H-RAS genetic alterations were mutations at hotspot codons 12, 13 and 61. We found one mutation in codon 63, 2 in codon 14 and a frequent polymorphism at nucleotide 81T→C in 19 samples. Cal-33, HSC-2 and BB49 cell lines were found to be resistant to cetuximab. Treatment of Cal-33, HSC-2 and BB49 cell lines with the PI3K inhibitor LY294002 either alone or in combination with cetuximab, led to a marked reduction of their viability. Immunoblotting analysis showed that treatment of cetuximab resistant cell lines with the PI3K inhibitor LY294002 +/- cetuximab resulted in marked decrease in levels of phospho-AKT. Xenograft experiments showed that tumor volumes were significantly reduced in LY294002 and LY294002 + cetuximab treated cell lines. Conclusions: These results indicate that the activation of RAS/PI3K signaling pathway is implicated in resistance to cetuximab in HNSCC. The functional significance of mutations in codons 14 and 63 and the 81T→C polymorphism needs to be determined.