Indiana University School of Dentistry Department of Endodontics / Introduction: Triple antibiotic paste (TAP, 1000 mg/ml) is composed of equal portions of ciprofloxacin, metronidazole and minocycline and is used as an intracanal dressing to disinfect the infected immature root canal during endodontic regeneration procedures. Lower concentrations of TAP have been recommended to minimize detrimental effects on pulp stem cells. TAP can be retained within the dentin matrix and its continual release confers an antibacterial effect to the dentin. Objective: The aim of this in vitro study was to investigate the residual antibacterial effect of dentin treated with various concentrations of TAP loaded into a gel system. Materials and Methods: Radicular dentin slabs were prepared from human teeth after obtaining IRB approval. The slabs were sterilized and treated with methylcellulose-based TAP of 100 mg/mL, 10 mg/mL, 1 mg/mL, 1.5% NaOCl, placebo paste with no TAP, or a positive control group with pure 1000 mg/mL TAP. Samples in each group were treated with the assigned TAP concentration for three weeks or immersed in 1.5% NaOCl for five minutes (n =18 per group). All samples were then irrigated with sterile water followed by 17% EDTA and incubated in phosphate buffered saline for either 2 or 4 weeks. Samples were then inoculated with Enterococcus faecalis and incubated for an additional 3 weeks. Biofilm formed on each sample was then dislodged and spiral plated to evaluate the bacterial colony-forming units. Data were analyzed using Fisher’s Exact tests and Wilcoxon rank sum tests (α = 0.05). Results: Dentin treated with 10, 100, or 1000 mg/mL of TAP demonstrated significant residual antibacterial effects up to four weeks. However, only 100 mg/mL TAP was able to completely prevent bacterial colonization after four weeks. No considerable residual antibacterial effect was observed in dentin treated with placebo gel, 1 mg/ml TAP or 1.5% NaOCl. Conclusion: At least 10 mg/mL of TAP loaded into a methylcellulose system is required to achieve a substantial residual antibacterial effect for four weeks.