We have examined the cytotoxic effect of gemcitabine in intravesical therapy using anin vitroco-cultured spheroid model composed of transitional cell carcinoma (TCC) and fibroblasts from both human and rat species. Immunohistochemistry analysis of the co-cultured spheroids, using cytokeratin-13 and vimentin antibodies against TCC and fibroblasts, respectively, showed the central location of fibroblasts within the spheroid, whereas TCC formed the peripheral layers. Spheroids composed of human TCC and fibroblasts (MGH-U3/CRL-1120 or RT-112/CRL-1120) as well as rat TCC and their corresponding fibroblasts (AY-27/RF-Ed1) displayed the same drug tolerance profile after an exposure of 0, 1, 3, 5, 7 and 14 days. As confirmed by time-lapse photography, MTT essay and vital dye staining, gemcitabine selectively killed the human and rat bladder cancer cell lines, but did not affect un-transformed human and rat fibroblast lines.