A new method for studying leakage of root fillings using the bacterium Pseudomonas fluorescens ATCC 13525 is described. The presence of the microorganism is detected by fluorimetry and can thus be used to measure the depth of penetration from the root apex toward the crown of the tooth. This system, applied to a number of methods of root canal filling, showed that procedures involving compaction of the gutta-percha gave a more effective seal than the use of a paste sealer with uncondensed gutta-percha. There was no statistically significant difference between the leakage results from the lateral, vertical, and thermomechanical condensed techniques.