Aim: To investigate the distribution of bacteria within the dentine tubular network in a two-chamber model in order to determine a possible route of penetration. Methodology: Root canals of teeth in the experimental group (n = 16) were instrumented and root filled using AH26 and gutta-percha. Canals in the teeth serving as positive (n = 1) and negative (n = 1) controls were instrumented, but not root filled. A two-chamber model was fabricated for each root - upper chambers were inoculated weekly with Streptococcus gordonii in brain-heart infusion broth over 90 days. Turbidity of the lower chamber was checked daily. Samples that showed turbidity during the experimental period and samples that showed no signs of turbidity at 90 days were fractured and prepared for SEM examination. SEM examination for the presence of bacteria within the dentinal tubules was performed in the cervical, middle and apical root thirds. In each root third, the depth of bacterial penetration was recorded as inner, middle or outer dentine. Results: Bacteria were most commonly detected in the cervical third (14 of 30 areas). Irrespective of the root third examined, bacteria were most commonly detected in the inner dentine (i.e. adjacent to the root canal). Only two samples demonstrated bacterial penetration in the outer dentine. Conclusion: The dentine tubular network provides a potential pathway for bacteria to penetrate tooth roots in a two-chamber model. © 2014 by The American College of Veterinary Surgeons.