The present study demonstrated the three-dimensional microstructure of the rat diaphragm by scanning electron microscopy (SEM) of either intact or alkali-treated tissues, enzyme-histochemistry, and confocal laser scanning microscopy (LSM). The peritoneal and pleural surfaces of the diaphragm were covered with mesothelial cells studded with microvilli. Many round gaps were formed between the mesothelial cells. The submesothelial connective tissue contained voluminous, irregularly shaped lymphatics. Some of these lymphatics extended many funnel-like projections of their endothelia towards the pored region of the mesothelium. On coming into contact with the mesothelium, many of the lymphatic projections were perforated at their ends, thus giving rise to stomata connecting the peritoneal cavity and lymphatic lumen. Some projections ended blindly while plugging the mesothelial pores, thereby making visible some intercellular gaps in this contact. The subperitoneal sheet of the collagen fiber network possessed clusters of foramina which tightly fit the passage of the lymphatic projections. Confocal LSM of the diaphragm after intraperitoneal injection of FITC-dextran demonstrated the tracer both in the lymphatic lumina and in the connective tissue spaces. Our results indicate that peritoneal fluid is allowed to flow into the connective tissue spaces of the diaphragm through intercellular gaps and into lymphatics through stomata.