The peritoneal mesothelium is composed of an extensive monolayer of mesothelial cells that lines the body's serous cavity and internal organs and was previously thought to act principally as a protective nonadhesive lubricating surface to facilitate intracoelomic movement. With the introduction of peritoneal dialysis over three decades ago, there has been much interest in the cell biology of peritoneal mesothelial cells. Independent studies have highlighted specific properties of the peritoneal mesothelial cell, including antigen presentation, regenerative properties, clearance of fibrin; synthesis of cytokines, growth factors, and matrix proteins; and secretion of lubricants to protect the tissue from abrasion, adhesion, infection, and tumor dissemination. It is now evident that the mesothelium is not merely a passive membrane but, rather, a dynamic membrane that contributes substantially to the structural, functional, and homeostatic properties of the peritoneum. Since peritoneal mesothelial cells in culture possess immunohistochemical markers identical to mesothelial stem cells, the culture of mesothelial cells offers researchers an essential tool to assess their morphologic, structural, and functional properties. This review will discuss current procedures to isolate peritoneal mesothelial cells from human omental specimens, animal sources, and spent dialysate. Furthermore, the functional and morphologic properties of mesothelial cells are discussed, together with the potential use of mesothelial cell culture in research and clinical applications.