The purpose of this in vitro study was to evaluate the accuracy of three apex locators in determining the working length during the retreatment process. Twenty extracted single-rooted human teeth with mature apices were used in this study. The root canal length of each tooth was measured placing a #15 file until the tip was visible at the apical foramen. The direct visual measurement was reduced by 0.5 mm and recorded. The root canals were instrumented and filled to the direct visual measurement using lateral compaction technique. After 7 days the teeth were retreated using three apex locators: ProPex, NovApex, and Root ZX, for determining the retreatment working length. Afterward, comparison between the visual working length and the retreatment working length were made. ProPex, NovApex, and Root ZX were accurate within 0.5 mm 80, 85, and 95% of the time, and within 1 mm 95, 95, and 100%, respectively. No significant differences were detected between the three apex locators (p > 0.05).