Background: Novel therapies are necessary to improve outcomes for patients with squamous cell carcinomas (SCC) of the head and neck. Historically, vaccinia virus was administered widely to humans as a vaccine and led to the eradication of smallpox. We examined the therapeutic effects of an attenuated, replication-competent vaccinia virus (GLV-1h68) as an oncolytic agent against a panel of six human head and neck SCC cell lines. Results: All six cell lines supported viral transgene expression (beta-galactosidase, green fluorescent protein, and luciferase) as early as 6 hours after viral exposure. Efficient transgene expression and viral replication (>150- fold titer increase over 72 hrs) were observed in four of the cell lines. At a multiplicity of infection (MOI) of 1, GLV-1h68 was highly cytotoxic to the four cell lines, resulting in >= 90% cytotoxicity over 6 days, and the remaining two cell lines exhibited >45% cytotoxicity. Even at a very low MOI of 0.01, three cell lines still demonstrated >60% cell death over 6 days. A single injection of GLV-1h68 (5 x 10(6) pfu) intratumorally into MSKQLL2 xenografts in mice exhibited localized intratumoral luciferase activity peaking at days 2-4, with gradual resolution over 10 days and no evidence of spread to normal organs. Treated animals exhibited near-complete tumor regression over a 24-day period without any observed toxicity, while control animals demonstrated rapid tumor progression. Conclusion: These results demonstrate significant oncolytic efficacy by an attenuated vaccinia virus for infecting and lysing head and neck SCC both in vitro and in vivo, and support its continued investigation in future clinical trials.