An in vitro model for studying the invasion mechanism of oral squamous cell carcinoma (SCC) was developed using a specific culture matrix composed of collagen gel combined with human fibroblasts. Five SCC cell lines cultured on collagen-only gels showed stratified growth on the gels. However, all five cell lines showed invasive growth into the matrix when cultured on the fibroblast-incorporated collagen gels. Moreover, fibroblast-conditioned medium was shown to promote the invasion of HSC-3 cells into the collagen gels. These results suggest that fibroblasts play an important role in the invasion of oral SCC cells in vitro. Four cell lines, which were newly established in our laboratory, were tested in this assay system. These cell lines cultured on fibroblast-incorporated collagen gels expressed morphologic and biologic characteristics in vitro, similar to those in vivo.