Purpose To investigate the potential of tissue-engineered pericardial (TEP) matrixes to form effective sources for in-vivo creation of urinary bladder wall grafts. Material and Methods Sixteen rabbits were divided into 4 groups. The control Group underwent classical autoaugmentation (Group 1). Other groups underwent insertion of 2x1cm2 frame acellular pericardium as a natural tissue expander between bladder mucosa and seromuscular layer. Group 2 underwent insertion of acellular pericardium. In group 3, inserted scaffold was implanted with autologous tissue fragments; biopsy of 10x5mm2 smooth muscle cell (SMC) layer of bladder was dissected and minced into 20 fragments. The fragments were coated on bladder mucosal layer. In group 4 the SMCs were isolated and cultured from dissected SMC layer and seeded on acellular pericardium. After 6 weeks, the in-vivo constructed muscular layer was grafted to remaining bladder host muscular layer. Before grafting and at 30d intervals after grafting, bladder biopsies were obtained for determination of CD31/34, SMC α-actin, and cytokeratin AE1/AE3 following cystometric evaluations. Results The frames containing fragment-seeded and cell-seeded tissue expanders, demonstrated more organized muscular wall generation with mature muscular layers, while number of CD34+ endothelial progenitor cells and CD31+ microvessels were significantly higher(P Conclusions The results demonstrate the bladder wall acted as an in-situ bioreactor; preserved autologous bladder tissue culture with seeded fragments and cells on a tissue expander provided a histologically organized muscular wall. Application of decellularized pericardium was an effective method for in-vivo constructed grafts in augmentation cystoplasty.