A simple method of preparing rat bladder epithelial cells for flow cytometry was developed, which involves the procedure of (1) eversion of urinary bladder, (2) dissociation of the urothelial cells from the underlying connective tissue using an enzyme cocktail (collagenase, dispase, and trypsin) and (3) subsequent treatment with EDTA. The use of trypsin was essential to remove erythrocytes from cell suspension. This method allowed us to obtain at least 5×105 cells per bladder which were sufficient to measure DNA content distribution more than 10 times per animal. Despite use of trypsin, this method yielded DNA histograms with a sharp G0/G1-peak whose coefficient of variation was approximately 5%. The percentages of cells in G0/G1, S, and G2/M phases (mean±standard deviation) were 96.0±1.3%, 0.5±0.1%, and 3.2±1.1%, respectively. Thus, the results indicate that this method in a good isolation procedure to obtain a number of single urothelial cells for flow cytometry.