It is assumed that above a critical level, microleakage causes periapical disease and jeopardizes periapical tissue repair. The purpose of this investigation was first to find out to what degree obturation prevents leakage of bacteria-sized particles or large protein molecules, and second if leakage of the commonly used dye methylene blue is comparable with that of a small bacterial metabolic product of similar molecular size. Sections, 9 mm long, obtained from 46 extracted human incisor roots had their root canals prepared in a standardized manner and obturated in four experimental ways using gutta-percha. The root sections were mounted in the middle of tubes that were closed at both ends with rubber membrane stoppers, which permitted sampling from the apical reservoir. The coronal reservoir was filled with a solution of 1 mg/ml latex beads, 4 micrograms/ml endotoxin, 0.5 per cent butyric acid, 0.1 per cent valeric acid and 0.1 per cent methylene blue water. After 1 and 2 weeks, samples were taken from the apical reservoir and tested for the presence of latex beads by phase-contrast microscopy, for endotoxin with a limulus lysate test, for butyric acid with gas-chromatography and for methylene blue with spectrophotometric analysis. Leakage of bacteria-sized particles and large-sized protein molecules could be prevented only when both sealer and pressure were used in obturating root canals with gutta-percha. Leakage of butyric acid proved to be comparable with leakage of methylene blue; microleakage of these small molecules could not be prevented in this study whatever method of filling was used.