The purpose of this study was to develop a method for the depletion of macrophages from the peritoneal cavity and the omentum of the rat. Rats received two intraperitoneal injections (at days 0 and 3) with liposome-encapsulated clodronate (dichloromethylene bisphosphonate: Cl2MBP-liposomes). This treatment resulted in complete elimination of mature tissue macrophages (ED2-positive macrophages) from the peritoneal cavity and the omentum within 2 days. The elimination included the strongly ED2-positive spindle-shaped cells of the omental membrane. Repopulation of the omental ED2-positive macrophages was not seen within the next 23 days. Whereas ED2-positive macrophages were completely depleted, few ED1-positive cells remained and repopulation of ED1-positive cells was faster. The treatment further depleted macrophages from the spleen, especially from the red pulp, parathymic lymph nodes and liver. Freund's incomplete adjuvant administered one day after the last injection of Cl2MBP-liposomes considerably accelerated repopulation in the omentum. The protocol described might be used to investigate the contribution of mature tissue macrophages to the induction of immune responses, drug metabolism and the elimination of intestinal tumours.