The roles of tumor stroma in carcinogenesis are still unclear. This study was aimed at designing an in vitro model for investigating the effects of stromal fibroblasts in the invasive growth of squamous cell carcinoma. Using two cancer cell lines, we performed three-dimensional co-culture with dermal equivalents to evaluate the effects of fibroblasts in cancer invasion. In vitro models for cellular interaction study were designed as follows: a collagen gel-based direct co-culture model (C-Dr) and a collagen gel-based indirect co-culture model (C-In). The invasive growth was found only in the dermal equivalents with fibroblasts. MMP-2 activity could be induced by direct contact between cancer cells and stromal fibroblasts. Cathepsin D was also highly expressed when co-cultured with cancer cells and fibroblasts. The present study demonstrated that the presence of fibroblasts is essential in cancer invasion and that collagen gel-based co-culture models might be useful for invasive study. (c) 2006 Elsevier Inc. All rights reserved.