In search of alternatives for urothelial-lined augmentation or reconstruction of the urinary bladder, this study combined the application of seromuscular gastrointestinal (GI) segments with the use of in-vitro cultured, autologous urothelial cells in a sheep model. A cell culture system was set up for establishment and expansion of urothelial cells out of small biopsies from bladder mucosa. A biodegradable carrier made of lactidcaprolactoncopolymer was introduced, allowing upside-down transplantation of cell cultures in vivo. Bladder mucosal biopsies were taken from 14 sheep (mean weight 13.3 kg) with an average yield of 3.5×10(5) viable cells/cm(2) after trypsinization. Primary low-density cultures grew to confluence within 5-7 days. Secondary cultures were established on the biodegradable film and were available a week later. They were transplanted onto demucosalized segments of stomach (group 1) or colon (group 2) in 5 animals each, followed by bladder incorporation in clam fashion. The earliest specimens, demonstrating survival and some proliferation of the cultured urothelium in both groups, were obtained 13 days postoperatively. To exclude urothelial regrowth, a temporary pouch grafted with cultured urothelium was created in 2 more sheep of each group. Biopsies were taken after 2 and 3 weeks, respectively, when the reopened pouch was integrated into the bladder (delayed augmentation). In these pouches, adherence and proliferation of urothelial cells could not be demonstrated. Limited radiologic and urodynamic investigations after 5-6-month follow-up revealed good shape, capacity, and compliance of the primarily augmented bladders only. The results indicate that urothelial cell cultures can be established and applied in vivo. Despite upside-down transplantation, they are able to survive on seromuscular segments in an autologous setting. The bladder environment is necessary to promote complete covering of the seromuscular segments. Based on our histologic findings, the share of both resident bladder urothelium and transplanted cells in the formation of the final urothelial lining remains uncertain. Morphologic and urodynamic follow-up data indicate that this process can be accelerated by the transplanted urothelial cells, reducing fibrotic changes of the GI segments. The functional quality of the augmented bladder seemed to improve compared to results after seromuscular augmentation alone.