Dialysate concentration of phospholipids has been used to monitor peritoneal membrane status. However, we recently found that the peritoneum has a surface layer in which phospholipids may be the main constituent. Therefore, in this study, we compared the phospholipids composition of peritoneal dialysate and of the peritoneal surface in rats. Eight male Sprague-Dawley rats were used in the study. Five rats received an intraperitoneal injection of 25 mL 4.25% glucose dialysis solution. After four hours, the rats were killed, and the dialysate was drained completely. Then 20 mL of Folch solution was infused into the peritoneal cavity for 30 seconds and drained completely. The other three rats received the Folch solution without dialysis. The effluent and Folch solution were then processed for phospholipids analysis using high performance thin-layer chromatography (HPTLC). The total phospholipids content was ten times higher in the surface layer than in the dialysate effluent. In the effluent, four clearly different components were seen: lysophosphatidylcholine (LPC), sphingomyelin (SM, 29%), phosphatidylcholine (PC, 66%), and phosphatidylinositol (PI, 4.5%). However, in the surface layer, as well as LPC, SM (20.6%), PHC (47%), and PI (6.3%), two additional components were seen, phosphatidylserine (PS, 17.1%) and phosphatidylethanolamine (PE, 8.9%). The quantity of phospholipids in the peritoneal surface of non dialyzed rats was similar to the total quantity of phospholipids (in effluent and in the peritoneal surface) of dialyzed rats. Our results suggest that: (1) a surface layer is present on the peritoneum; this layer could well be extracted by Folch solution; and, with appropriate incubation time, one can separate the surface layer without damaging the mesothelial cells; (2) the composition of phospholipids in the effluent is different from that in the peritoneal surface layer, which contains membrane phospholipids (PS and PE); (3) shielding from the peritoneal surface may be the main reason for the presence of phospholipids in the dialysate.