The purpose of this study was to distinguish passive retention of microbubbles in liver sinusoids from active intracellular retaining, and to provide further evidence of post vascular liver specific phase of perflubutane microbubble contrastenhanced ultrasound for potential use in the detection of focal liver lesions. Fifty-four rats were divided into three groups: saline group (G1, as a control, n = 6), sulphur hexafluoride microbubble group (G2, n = 24) and perflubutane microbubble group (G3, n = 24). The livers were perfused at 2, 5, 10 and 20 min after injection of perflubutane or sulphur hexafluoride gas-filled microbubbles or normal saline. Changes in contrast-enhanced images within the liver were quantified. After perfusion, the echogenicity of the liver in the saline group increased from −49.44 ± 0.08 dB to −44.37 ± 1.02 dB (p < 0.05). In G2, the enhancement decreased significantly after perfusion at 2 and 5 min, and increased at 10 and 20 min (all p < 0.05). In G3, the enhancement decreased from −18.05 ± 2.28 dB to −26.76 ± 1.69 dB only at 2 min after perfusion (p < 0.05). Perflubutane microbubbles provided a post vascular liver specific phase on contrast-enhanced ultrasound, which could begin as early as 5 min after administration of the contrast agent in rats. This study suggests that perflubutane microbubbles could result in a liver specific phase and provide valuable information for diagnosis and detection of focal liver lesions which may improve the efficiency of contrast-enhanced ultrasound in clinical diagnoses of liver diseases.