The peritoneum lines the abdominal cavity, covering and supporting the abdominal organs. The layer of mesothelial cells provides a functional barrier and allows vectorial transport between serous fluid of the abdominal cavity and tissue fluid. Based on the anatomical localization, three peritoneal regions can be distinguished: parietal, visceral and diaphragmatic. However, a comparative analysis of their barrier and transport properties has not yet been carried out. Electrophysiological parameters of three different regions of the rat peritoneum were investigated here using the Ussing chamber. The parietal peritoneum revealed the highest transmesothelial potential (2.2 ± 0.3 mV), short circuit current (19.8 ± 1.7 мA/cm2), and transmesothelial resistance (94.9 ± 3.5 Ohm cm2) compared to other peritoneal regions. The addition of ouabain (1 mM) from the apical and basolateral sides of the parietal and visceral peritoneum resulted in an increase in the transmesothelial resistance. In addition, a histological analysis was performed. Tissue preparations of the parietal peritoneum comprised a layer of mesothelial cells and adjacent striated muscle fibers with small interlayers of loose connective tissue. Tissue specimens of the diaphragmatic and visceral peritonea included two layers of mesothelial cells. In the diaphragmatic peritoneum, they were separated by muscle fibers and large areas of loose connective tissue, while in the visceral peritoneum by adipose and connective tissues. In conclusion, the parietal and visceral regions of the rat peritoneum contribute differentially to transmesothelial transport. The parietal peritoneum exhibits pronounced barrier and transport properties and can be considered as a promising model for studying the molecular interaction between Na/K-ATPase and tight junction proteins, claudins.