Three-dimensional multicellular spheroids of two fibroblast cell lines (WI-38 and N1) and two differently differentiated bladder carcinoma cell lines (RT4 and J82) were used for cocultures of multicellular tumor spheroids with multicellular spheroids of fibroblasts. The aim of the study was the establishment and characterization of a standardized three-dimensional model for studies of tumor cell-fibroblast interaction as one aspect of tumor-stromal cell interactions of in vivo tumor tissue. Interaction of multicellular spheroids of both fibroblast cell types was analyzed by staining with antibodies against cytokeratin, vimentin and different extracellular matrix molecules. Further, proliferation assessment and phenotypic characterization of the cocultures are presented. Interactions varied with tumor cell type and fibroblast cell type, reflecting intrinsic properties of tumor cells and fibroblasts. The coculture of tumor cells with N1 reflected the in vivo situation the closest, since invasive properties of J82 as well as noninvasive properties of RT4 were characteristics seen in coculture.