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Background: Adenomyomatosis of gallbladder is an acquired hyperplastic lesion, characterized by focal or diffuse thickening of the gallbladder with intramural cysts or echogenic areas with comet tail on ultrasonography. But in some cases, especially in the localized fundal type of adenomyomatosis, the intramural anechoic cystic spaces are uncertainty which causes difficult to differential adenomyomatosis from GB cancer. The purpose of this study was to determine the accuracy of real-time contrast-enhanced ultrasonography(CEUS) in the diagnosis of the fundal localized type of gallbladder adenomyomatosis., Methods: We performed a retrospective study of 21 patients with pathologically proven fundal localized type of gallbladder (GB) adenomyomatosis. All patients underwent preoperative grayscale ultrasound (US) and real-time CEUS examination. The study's reviewers made the diagnosis of adenomyomatosis according to the presence of the focal thickening of the fundal gallbladder wall with intramural cyst or intramural echogenic foci on grayscale US or CEUS. The diagnostic accuracy of US and CEUS was compared. The enhanced pattern and degree of intactness of the GB wall were also recorded., Results: The fundal portion of the GB wall showed localized thickness in all 21 patients. Small anechoic spaces or intramural echogenic foci were detected in 14 (66.7%) and 21 (100 %) of cases respectively, and the intactness of the GB wall's outer hyper-echoic layer was demonstrated in 17 (81%) and 20 (95%) on grayscale US and CEUS, respectively. The accuracy rate of the above two examination modalities was significantly different (p < 0.05). In the arterial phase of the CEUS, areas of focal thickened GB wall were iso-enhanced in 18 cases and hyper-enhanced in 3 cases. All 21 cases appeared to show heterogeneous enhancement with small non-enhancement spaces. The mucosal and serosal layers of the GB wall surrounding the lesions were enhanced, which presented as two "hyper-echoic lines" in the arterial phase of CEUS. In the venous phase of the CEUS, 19 lesions were iso-enhanced and 2 lesions were hypo-enhanced. The small non-enhancement spaces were more clearly during the venous phase., Conclusion: The small non-enhancement space is a characteristic finding of the fundal localized type of gallbladder adenomyomatosis on CEUS. CEUS could increase the degree of visualization of Rokitansky-Aschoff sinuses (RAS) and intactness of the GB wall, which play an important role in differential diagnosis.