The purpose of this study was to determine the feasibility of using the pericardium as a source of endothelial cells. Nineteen pieces of fresh pericardium were obtained from nine mongrel dogs. Cells were prepared by collagenase digestion of the pericardium for 24 minutes followed by centrifugation. The cells were divided into three groups: The supernatant subjected to no further steps, Group I (N = 6); filtration through a 15 micron porous mesh, Group II (N = 6); and Percoll gradient separation with medium 199, Group III (N = 7). The cells obtained were cultured for seven days in tissue culture media. Yield (cells x 10(5)/gram fresh tissue) was determined with Methods I, II, and III, producing 32.4 +/- 25.9 (SD), 0.96 +/- 0.6 and 0.57 +/- 0.5, respectively (I vs II or III, p less than 0.01). Fibroblast contamination determined by phase contrast light microscopy was demonstrated in 6/6 cultures with Method I, 3/6 with II and 1/7 for III (I vs III, p less than 0.01). An assay for endothelial cells (Factor VIII) was positive in 2/6 cultures with Method I, 5/6 with II and 7/7 for III (I vs III, p less than 0.01). The pericardium is a suitable organ for procurement of endothelial cells. Though reducing yield, filtration and Percoll gradient separation allows for isolation of a relatively pure culture of endothelial cells.