Clinical Relevance Pigments in tooth structures affect the diffusion of H 2 O 2 through enamel and dentin. The bleaching methodology can be impacted. SUMMARY Objective: The aim of this study was to evaluate the influence of the presence of pigments in tooth structures on the trans-enamel and trans-dentin diffusion of hydrogen peroxide (H 2 O 2 ) and its cytotoxicity after carrying out an in-office bleaching therapy. Methods and Materials: A bleaching gel with 35% H 2 O 2 was applied for 45 minutes (three times for 15 minutes) on enamel and dentin discs (n=6), either previously submitted to the intrinsic pigmentation protocol with a concentrated solution of black tea, or not, defining the following groups: G1, unbleached untreated discs (control 1); G2, unbleached pigmented discs (control 2); G3, bleached untreated discs; G4, bleached pigmented discs. The discs were adapted to artificial pulp chambers, which were placed in wells of 24-well plates containing 1 mL culture medium (Dulbecco’s modified Eagle’s medium [DMEM]). After applying the bleaching gel on enamel, the extracts (DMEM + components of bleaching gel that diffused through the discs) were collected and then applied on the cultured MDPC-23 odontoblast-like cells. Cell viability (methyl tetrazolium assay and Live & Dead, Calcein AM, and ethidium homodimer-1 [EthD-1] probes), the amount of H 2 O 2 that diffused through enamel and dentin (leuco-crystal violet product), and the H 2 O 2 -mediated oxidative cell stress (SOx) and components of degradation were assessed (analysis of variance/Tukey; α=0.05). Results: There was no significant difference between the groups G1 and G2 for all the parameters tested ( p >0.05). Reduction in the trans-enamel and trans-dentin diffusion of H 2 O 2 occurred for G4 in comparison with G3. Significantly lower cell viability associated with greater oxidative stress was observed for G3 ( p <0.05). Therefore, in-office tooth bleaching therapy performed in pigmented samples caused lower cytotoxic effects compared with untreated samples submitted to the same esthetic procedure ( p <0.05). Conclusion: According to the methodology used in this investigation, the authors concluded that the presence of pigments in hard tooth structures decreases the trans-enamel and trans-dentin diffusion of H 2 O 2 and the toxicity to pulp cells of an in-office bleaching gel with 35% H 2 O 2 .