Introduction. In order to exclude the difficulties arising in the course of traditional surgical interventions in the scope of substitutional urethroplasty, in recent years, alternative materials have been developed using tissue engineering. This study is devoted to the development of new tissue-engineered constructions for urethroplasty using cells of different tissue origins and biopolymers, which is an urgent problem of modern medicine. Aim. Experimental provision of a rationale for the possibility of using tissue-engineered constructions to replace urethral defects. Methods and materials. Two-staged, experimental, controlled study. Dedicated to the development of tissue-engineered constructions (TEC) based on biopolymers seeded with mesenchymal stem cells or buccal epithelial cells. The prepared TECs were implanted into the rabbits urethra wall in a acute trauma model. Comparison of the results of the TECs usage with the gold standard buccal urethraplasty was carried out. Results. Urethrography showed similar results in all groups of animals, regardless the type of implanted material. No urethral patency was found, and confocal microscopy of urethral cryosections revealed the presence of nanoparticle-labeled mesenchymal stem cells / buccal epithelium cells with signs of their differentiation in the urothelial direction in the mucous layer. Conclusion. The possibility of using tissue-engineered constructions based on biopolymers containing autologous mesenchymal stem cells or buccal epithelium cells for experimental substitutional urethroplasty was shown. The developed TECs can be used as an alternative to buccal urethroplasty in an experiment.