In the present study, we evaluated the efficacy of bleaching on enamel and opposite dentin surfaces using 35% hydrogen peroxide (HP) with the prior application of a desensitizing agent. Thirty bovine dental fragments, with thicknesses of 1 mm enamel and 1.75 mm dentin, were stained in a solution of black tea. The fragments were randomly divided into three groups (n = 10) according to the following protocols: (a) 2% neutral fluoride + bleaching; (b) desensitizing agent + bleaching; and (c) without desensitizer + bleaching. The bleaching efficacy was evaluated four times: after staining with tea (baseline) and after each of the 3 weeks of bleaching, by means of the CIE Lab method using a reflectance spectrophotometer. The data coordinate L* was evaluated by an analysis of repeated measures with PROC MIXED and Tukey-Kramer's test. The ΔE values were subjected to anova and Tukey's test (α = 0.05). The 35% HP treatment showed greater efficacy on deep dentin after removal of the enamel stains, with increasing means during all times in all treatments. The use of a desensitizing agent prior to the bleaching session did not affect the mechanism of action of 35% HP with regard to tooth depth.