BACKGROUND The aim of this study was to investigate the substantivity of chlorhexidine (CHX) solution and gel within a root canal system for 24 hours, 30 days, and 90 days. METHODS Forty-five extracted human anterior teeth were used for this study. The samples were divided into 3 groups according to the chemical auxiliary substance used to perform the root canal preparation: group 1, 2% liquid CHX; group 2, 2% gel CHX; and group 3, distilled water (the control group). The working length was determined by inserting a #10 K-file into the canal up to the moment its tip was seen in the apex foramen and then withdrawing it 1 mm. The roots were prepared up to the instrument #45. Longitudinal grooves were carved on the free surfaces of the roots, providing 2 halves of each root and resulting in 30 samples per group. Each group was randomly divided into 3 subgroups (n = 10), and substantivity was evaluated after 24 hours, 30 days, and 90 days of incubation. The amount of CHX (in micrometers) was measured through reverse-phase high-performance liquid chromatography. Statistical analysis was performed by analysis of variance and the Tukey test for post hoc comparisons (α = 0.05). RESULTS The control group showed no substantivity. Significant amounts of CHX solution and gel remained retained in dentin substrates independent of the time of incubation (P < .05). CHX solution showed a higher substantivity than CHX gel, with the exception of groups incubated for 90 days. The decreasing amounts of retained CHX inside the canal were for 24 hours >30 days >90 days for CHX solution and 24 hours >30 days ≥ 90 days for CHX gel. CONCLUSIONS The results of this study indicate that CHX solution and gel are retained in root canal dentin for up to 90 days.