Invasion and metastasis in oral squamous cell carcinoma (OSCC) are associated with changes in the extracellular matrix (ECM). We have previously shown an extracellular co-deposition of laminin-5 (Ln-5) and large unspliced tenascin-C (Tn-C(L)) in OSCC. Using a co-culture model of hTERT-BJ1 fibroblasts and the OSCC cell line PE/CA-PJ15, we demonstrate in the present study that Ln-5 and Tn-C(L) are not only co-deposited, but also form a physical complex which can be recovered by co-immunoprecipitation. In agreement with these results, examination of OSCC tissue specimens of different malignancy grade by means of confocal laser scanning microscopy revealed different patterns of Ln-5 and Tn-C(L) co-localization implicating complex formation also in vivo. A ribbon like co-localization was detected in subepithelial basement membranes around well differentiated OSCC parts and tumor clusters. Furthermore, a fibrillar Ln-5 gamma2 chain/Tn-C(L) co-localization occurred in the carcinoma stroma beneath tumor clusters. Additionally, at the site of ruptured basement membranes there were dot or strand like co-deposits of both molecules, but co-localizations were only rarely detectable. These different patterns may reflect a sequential modulation and reorganization of the ECM in the tumor/stroma interface as it occurs in different stages of OSCC invasion.