Canine peritoneal macrophages were obtained by repeated peritoneal lavage of dogs at 14 day intervals. Administration i.p. of sterile mineral oil increased the leukocyte yield approximately 20-fold and the macrophage recovery approximately 35-fold. Cell recovery was maximum 7-21 days after administration of oil and declined slightly by 35 days. Restimulation with a 2nd injection of oil promptly revitalized cell recovery. Peritoneal macrophage cultures were well established by 6 days after seeding and were maintained for at least 30 days. Initially, cultured macrophages were mononuclear, but binuclear cells appeared after 6 days'' cultivation and multinucleated giant cells were observed after 14 days. Canine peritoneal macrophages and peripheral blood monocytes were equally susceptible to infection with E. canis. Infected cells were detected by 60 h after inoculation, and replication was evident by 12-18 days.