Recent studies indicate that the specificity of p38 mitogen-activated protein kinase (MAPK)-mediated cellular stress responses is determined by the expression pattern of the distinct p38 isoforms. Here, we have analysed the function of distinct p38 isoforms in the growth and invasion of head and neck squamous cell carcinomas (HNSCCs). Activation of p38 MAPK by arsenite resulted in inactivation of the ERK1, 2 signaling pathway by dephosphorylation of MEK1, 2 in primary human epidermal keratinocytes (HEKs), whereas in HNSCC cells this p38-mediated inhibition of the ERK1, 2 pathway was absent. Quantitation of p38 pathway component mRNA expression in HNSCC cell lines (n= 42) compared to HEKs (n= 8) revealed that p38α and p38δ isoforms are predominantly expressed in both cell types and that MKK3 is the primary upstream activator expressed. Inhibition of endogenous p38α or p38δ activity by adenoviral delivery of corresponding dominant-negative p38 isoforms potently reduced MMP-13 and MMP-1 expressions, and suppressed the invasion of HNSCC cells through collagen. Dominant-negative p38α and p38δ inhibited squamous cell carcinoma (SCC) cell proliferation and inhibition of p38α activity also compromised survival of SCC cells. p38α and p38δ were predominantly expressed in HNSCCs (n= 24) and nonneoplastic epithelium in vivo (n= 6), with MKK3 being the primary upstream activator. Activation and expression of p38α and p38δ by tumor cells was detected in HNSCCs in vivo (n= 16). Adenoviral expression of dominant-negative p38α or p38δ in cutaneous SCC cells potently inhibited their implantation in skin of severe combined immunodeficiency mice and growth of xenografts in vivo. Our results indicate that p38α and p38δ specifically promote the malignant phenotype of SCC cells by regulating cell survival, proliferation and invasion, suggesting these p38 MAPK isoforms as potential therapeutic targets in HNSCCs.