Endotoxin, elaborated by gram-negative organisms, is an important factor in apical periodontitis. The objective of this study was to evaluate the magnitude of endotoxin penetration through root canal treated teeth using a dual chamber model system. Forty-four maxillary anterior teeth were prepared endodontically and canals filled either by lateral condensation or a warm thermoplasticized technique in combination with either Roth's 801 or AH 26 sealer. Teeth were suspended in the model system with a mixed anaerobic bacterial suspension in the upper chamber and HBSS in the lower chamber. The QCL-1000 LAL assay was used to measure endotoxin at 0, 1, 7, 14, and 21 days. Response feature analysis using trapezoidal area under the curve was performed; the four treatment groups were compared using nonparametric methods. Groups differed (p = 0.028), with thermoplasticized root canal filling/Roth's 801 sealer permitting the least apical endotoxin penetration. Results suggest that Roth's 801 sealer may have a role in inhibiting endotoxin penetration.