Here we evaluate an improved orthotopic rat bladder tumor model, to be used for the evaluation of the therapeutic potential of novel cancer therapeutics. Before instilling AY-27 tumor cells into chemically denudated rat bladders, AY-27 cells were labeled with the fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI). We found that the presence of Dil did not alter the in vitro AY-27 cell proliferation and that the Dil label was strongly associated with the cells. We further provide evidence that the use of fluorescently labeled AY-27 tumor cells allows the visualization and hence the validation of the orthotopic tumor inoculation process. Using this technique it was possible to track down the tumor cells after inoculation into the bladder, which makes it straightforward to distinguish tumor cells from remaining or regenerated normal urothelium over a period of 5 d. The results also demonstrated that malignant AY-27 tissue exists as an intact non-muscle invasive bladder tumor only for 1-3 d after cell implantation. Accordingly the AY-27 bladder tumor model was used to evaluate the antitumoral effect of a single intravesical MM-C instillation. All rats instilled with 1 mM MM-C survived the final endpoint of 30 d after intravesical MM-C. Moreover, 10 and 30 d after treatment the urothelium of the MM-C-treated animals was completely restored. Remarkably, after MM-C treatment distinct patchy fluorescent dots were found into the submucosa and the regenerated urothelium, suggesting that dye retention is secondary to the digestion of Dil-loaded AY-27 cells and cellular debris by macrophages and related immune cells.