Third-generation electronic apex locators for root canal length determination are very reliable and are not subject to interference from the contents of the canals. This study compared the effectiveness of different embedding media for in vitro assessment of electronic apex locators. The tooth lengths of 30 extracted human mandibular central incisors were measured by introducing a size 15 K-file fitted with a silicone stop into the canal until its tip appeared through the apical foramen; the distance between the tip and stop was measured. The teeth were placed in cylindrical polyethylene tubes filled with different embedding media (1% agar, gelatin, alginate, saline, and flower sponge soaked in saline), and electronic reading was performed with the Root ZX device. Data were statistically assessed by the analysis of variance (ANOVA) and Tukey tests at a significance level of 5%. Despite the lack of statistically significant difference among the media, the flower sponge was the only medium in which the file surpassed the apex in some measurements.