Background: The use of electronic apex locators for working length determination eliminates many of the problems associated with the radiographic measurements (interference of anatomical structures, errors in projection such as elongation or shortening, and lack of three-dimensional representation). Its most important advantage over radiography is that it measures the length of the root canal to the apical constriction, not to the radiographic apex. The aim of this study was to assess the accuracy of a new fifth generation apex locator (Joypex 5) in recording the apical constriction and comparing it with a third generation apex locator (Root ZX) in vitro. Materials and method: Twenty four single-rooted sound human premolars, extracted for the purpose of orthodontic treatment and with fully-formed roots, were used in this study. Endodontic access cavity was prepared in each tooth and canal patency up to the apical foramen was checked with a #15 stainless steel K-file. No root canal preparation was performed. Root canal length measurement was done directly and electronically using two apex locators (Joypex 5 and Root ZX). Direct measurement of the root canal length was done by introducing a #15 K-file inside the root canal until its tip was just visible at the apical foramen, then removed from the root canal and its length was measured (in mm) and subtracted by 0.5 mm. For electronic measurement, the teeth were fixed in a sponge soaked in saline and the root canals were also filled with saline. The lip electrode was attached to the sponge and the apex locators were used according to the manufacturers' instructions. The file holder was clipped to the metal shaft of a #15 K-file and the file was then inserted inside the root canal and advanced until the display reading on the LCD of the apex locator was "0.5". The file was then removed from the root canal and its length was measured (in mm). The differences between the readings of each apex locator and the actual length of each canal were computed, and the results were analyzed statistically by paired t-test using SPSS Version 13. Results: The results of this study showed that the Joypex 5 apex locator showed a lower mean difference than the Root ZX apex locator as compared with the actual length, which was statistically significant (p<0.05). Concerning the accuracy of the two apex locators, Joypex 5 apex locator recorded the apical constriction exactly in 67%, while the Root ZX apex locator in only 25%. Within ±0.5 mm from the actual length, the accuracy of the Joypex 5 and the Root ZX were 83% and 67%, respectively. Within ±1 mm from the actual length, the accuracy of the Joypex 5 and the Root ZX were 100% and 96%, respectively. Conclusion: The Joypex 5 apex locator which is a fifth generation apex locator was more accurate in recording the apical constriction as compared with the Root ZX apex locator which is a third generation apex locator.