We have devised a bioreactor to simulate normal urinary bladder dynamics. The design permits a cell-seeded scaffold made from a modified porcine acellular matrix to be placed between 2 closed chambers filled with culture medium and be mechanically stimulated in a physiologically relevant manner. Specifically designed software increased hydrostatic pressure from 0 to 10 cm of water in a linear fashion in 1 chamber, resulting in mechanical stretch and strain on the scaffold. Pressure was increased over 55 min (filling) and then decreased to 0 over 10 s (voiding). Commercially available small intestinal submucosa scaffolds were used to test the mechanical capabilities of the bioreactor, and pressure waveforms were generated for up to 18 h. Scaffolds were seeded with bladder smooth muscle or urothelial cells and incubated in the bioreactor, which generated pressure waveforms for 6 h. Scaffold integrity was preserved as seen through Masson's trichrome staining. No obvious contamination of the system was noted. Hematoxylin and eosin staining showed presence of cells after incubation in the bioreactor, and immunohistochemistry and real-time reverse transcriptase polymerase chain reaction suggested continued cellular activity. Cellular orientation tended to be perpendicular to the applied pressure. Preliminary results suggest that our bioreactor is a suitable model for simulating normal physiological conditions of bladder cycling in an ex vivo system.