Intracellular depot formation may be an important component of the mode of action of doxorubicin (DXR)-containing liposomes. In this paper it was investigated whether it is possible that DXR is released from macrophages which have taken up DXR-containing liposomes in vivo. Macrophages were harvested from the peritoneal cavity of LOU/M rats after i.p. administration of DXR-liposomes. Two different liposome types were used for this investigation. The amount of DXR associated with macrophages was determined by high performance liquid chromatography. In order to monitor DXR release from the macrophages, an in vitro tumor cell growth inhibition assay was applied. Peritoneal macrophages collected 24 h after an i.p. injection of DXR-liposomes (10 mg/kg body weight) caused considerable growth inhibition of tumor cells in culture. The cytostatic potential of macrophage monolayers in vitro depended on the type of injected DXR-liposomes and was directly related to the amount of macrophage-associated DXR. The DXR content of the macrophage monolayers was completely released from the cells into the culture medium during the cocultivation with tumor cells. Supernatants obtained from monolayers, which were cultivated in the absence of tumor cells, showed a high growth-inhibitory activity. DXR either free or in liposomal form was chemically stable for up to 26 h during incubation with lysosomal fractions isolated from rat liver homogenates. The results indicate that release of DXR from macrophages which have phagocytosed DXR-liposomes in vivo is a real possibility.