The purpose of the study was to compare several methods that have been used to assess marginal leakage of root canal fillings. Sixty-three extracted, single-rooted teeth were instrumented and filled in a standardized manner. Teeth were randomly divided into groups of twenty and exposed to solutions containing methylene blue dye, calcium-45, carbon-14-labeled urea, and iodine-125-labeled albumin for 48 hours to compare the degree of leakage indicated by each technique. Methylene blue dye was found to penetrate farther up the canal than any of the isotope tracers. Carbon-14-labeled urea penetrated farther than the calcium-45- or iodine-125-labeled albumin. The mean volume of solution penetrating the teeth was exceedingly small (0.0011 ml) and probably unimportant physiologically.