Head and neck cancers are characterized by a vigorous desmoplastic response, but the contribution of stromal-derived growth factors to the tumor microenvironment is poorly understood. We evaluated the expression of stromal growth factor expression in head and neck squamous cell carcinoma (HNSCC) in normal and tumor-associated stromal cells. Stromal tissue was isolated from epithelial cells with laser capture microdissection (LCMD) and analyzed by cDNA array for the expression of TGFα, TGF-β1, HGF, PDGF-α, IGFII, bFGF, aFGF, VEGFC, and VEGF. Primary fibroblasts were isolated in vitro from HNSCC tumors, adjacent histologically normal mucosa, and skin in vitro. Fibroblast populations were assessed for TGF-β1 expression by ELISA and luciferase reporter assay to assess protein expression. We identified TGF-β1 and IGFII overexpression in normal and tumor-associated stromal cells; however, only TGF-β1 was significantly overexpressed (3.4-fold) in tumor-associated stroma. Assessment of carcinoma-associated fibroblasts (CAFs), normal dermal fibroblasts (NDFs), and normal mucosal fibroblasts (NMFs) in propagated fibroblasts demonstrated persistently elevated levels of TGF-β1 in CAFs compared to NMF and NDF populations. Elevated levels of TGF-β1 were identified in the stromal compartment of HNSCC tumors compared to normal mucosa by immunohistochemical analysis. These results suggest that TGF-β1 mRNA and protein is specifically upregulated in CAFs in vitro and in vivo. © 2004 Wiley-Liss, Inc.