Delayed parenchymal phase images of the liver more than 5 min after IV injection of ultrasound contrast agents are thought to be related to the phagocytosis of contrast agent microbubbles by macrophages. In this study, we examined whether liver-specific macrophages, Kupffer cells, phagocytosed the microbubbles and whether their elimination affected the delayed parenchymal images of the liver. Phase-contrast microscope observations showed that Kupffer cells phagocytosed various contrast agents in vitro. Among the contrast agents used, 99% of Sonazoid and Optison, and 47% of Levovist were phagocytosed, whereas only 7.3% of SonoVue and 0% of Imavist were phagocytosed. Elimination of Kupffer cells in vivo by gadolinium chloride (GdCl(3)) resulted in decreased intensity of the delayed parenchymal images with Sonazoid and Levovist, while SonoVue showed no changes compared with control. Our findings suggested that Kupffer cells phagocytosed contrast agents and they were responsible for the delayed images of contrast ultrasound in the liver.