whether C. sinensis infection is the etiological factor for cholecys- tolithiasis. Due to the subjectivity inherent in morphological analysis by light microscopy, we used real-time fluorescence PCR to detect the presence of C. sinensis eggs. The cytochrome C oxidase subunit 1 gene, a mitochondrial gene in C. sinensis present in hundreds or thousands of copies per cell, is more highly conserved than the ITS-2 sequences of C. sinensis and thus may be a good target for the development and evaluation with real-time PCR [41,42]. A real- time PCR assay based on the cytochrome coxidase subunit 1 gene of C. sinensis was previously used to successfully detect C. sinensis DNA in stool samples of rats [43,44]. Therefore, in the present study, we chose the cytochrome C oxidase subunit 1 gene as the target gene for real-time PCR detection, and the results confirmed that the eggs in the gallbladder stones were indeed C. sinensis eggs. To further explore the relationship between C. sinensis eggs and gallbladder stones formation without destroying the original structure of the stones, we analyzed the stones by SEM. SEM analysis revealed the presence of a large number of bilirubinate granules and a small amount of calcium carbonate crystals in the egg-positive gallbladder stones. Meanwhile, many of the C. sinensis eggs with a textured surface were discovered in gallbladder stones, and many tiny bilirubinate granules were adhered to the muskmelon wrinkles. Some eggs were wrapped with mucoid matter, while some were depressed or with thickened shell and/or without operculum. Other eggs displayed a shedding texture. The morphologic changes and diversity in the eggs might result from the extended periods that they had existed in the stones, or from nutritional deficiencies, calcification, nucleation and other factors. 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