Signal transducer and activator of transcription (STAT) proteins are constitutively activated in many malignancies, including squamous cell carcinoma of the head and neck (SCCHN). Previously, we reported that phosphorylation of the epidermal growth factor receptor (EGFR) is linked to activation of STATs 3 and 5 in SCCHN cells. The present study was undertaken to determine the role of Src family kinases in STAT activation and SCCHN growth. The Src family kinases c-Src, c-Yes, Fyn, and Lyn were expressed and activated by transforming growth factor-alpha stimulation in all four SCCHN cell lines examined but not in corresponding normal epithelial cells. In nine SCCHN cell lines tested, Src phosphotyrosine expression levels were highly correlated with activation levels of STATs 3 and 5. Co-immunoprecipitation analysis demonstrated interaction between c-Src and STATs 3 or 5 and EGFR in SCCHN cells, but no heterodimerization was detected between STAT3 and STAT5. SCCHN cells treated with either of two Src-specific inhibitors or transfected with a dominant-negative c-Src construct demonstrated decreased activation of STATs 3 and 5 and reduced growth rates in vitro. These results demonstrate a role for Src kinases in mediating activation of STATs 3 and 5 in concert with the EGFR in SCCHN cells. Strategies to target Src activation may contribute to the treatment of cancers that demonstrate increased levels of EGFR and STATs, including SCCHN.