Due to the problems encountered with the use of functionally different types of tissues for reconstructive urological surgery such as bladder, the use of regenerated autologous tissue is considering as a good alternative. Smooth muscle cells as the main contractile units of bladder wall are very important in preparation of engineered tissues for bladder replacement. The purpose of this study was isolation and propagation of smooth muscle cells from surgical specimens obtained from patients undergoing lower urinary tract open urological procedures. Surgical samples were collected with informed consent of patients undergoing open urological procedures. Samples were transported to the laboratory in transport medium .The bladder smooth muscle cells were cultured by the tissue abeling technique. Briefly the specimens were cut into small pieces. Fat and connective tissues were removed. Urothelium was detached and remaining stroma was minced into multiple small pieces. Then distributed evenly onto cell culture plates and DMEM, supplemented with 20% FBS, antibiotics and amphotericin B were added to the plates. The cultured tissues were maintained in a humidified 5% CO2 atmosphere at 37°C.The muscle cells were expanded until 70% confluence. Migration of the cells from stromal explants was apparent within one week. The cells showed spindle –shaped morphology and lack of contact inhibition at confluence.Immunoperoxidase abeling of cultured smooth muscle cells revealed α-actin expression by a group of the cells. Another type of the cells were seen which their morphology were different from spindle-shaped α-actin smooth muscle cells and seem to be smooth muscle myosin. Further investigation is needed to characterize them. Efficiency of isolation and propagation of the cells in vitro is very important in regeneration of autologous tissue for reconstructive surgery. On the other hand, shortage of donor tissue has made the investigators to harvest efficiently surgical materials as much as possible. Bladder muscle cells can be harvested from patients cultured and expanded in vitro to return into their body for reconstructive purposes. In this study we got experience to harvest urological surgical samples and propagate their smooth muscle cells .This experience together with another work on isolation and culture of human urothelial cells will help us to work more seriously on regeneration of autologous tissues for the patients.