PURPOSE: The aim of the present study was to define endosonographic criteria for an internal opening of fistulain-ano. METHODS: 118 patients with a diagnosis of fistulain-ano and a control group of 201 cases underwent endosonography in an outpatient setting. Confirmation of an internal opening was performed by intraoperative procedures. RESULTS: Of the 139 cases of fistulain-ano, the internal opening of 130 cases was confirmed. Analysis of endosonographic findings of the confirmed cases revealed that 122 cases showed positive findings for predictive location of an internal opening, and 8 cases demonstrated negative findings. Fifty-seven cases showed an appearance of a root-like budding formed by the intersphincteric tract that contacts the internal sphincter, classified as Criterion I. Fifty cases demonstrated the appearance of a root-like budding with an internal sphincteric defect, classified as Criterion II. Fifteen cases showed a subepithelial breach connecting to the intersphincteric tract through an internal sphincteric defect, classified as Criterion hi. Using the combination of these three criteria, the accuracy was as follows: sensitivity, 94 percent; specificity, 87 percent; positive predictive value, 81 percent; negative predictive value, 96 percent. CONCLUSION: These combined criteria would be of particular help in performing preoperative location of an internal opening with endosonography.