Bleaching systems and thickening agents lead to changes in the tooth enamel matrix as a result of an unspecific oxidation of the bleaching gel in the enamel. This reaction may result in a loss of mineral content and a decrease in microhardness. The aim of this study was to evaluate the effects of different bleaching systems and their thickening agents on the microhardness of human enamel in situ. Two dental slabs (3 x 3 x 2 mm) obtained from third molars were fixed on the buccal facial aspects of the maxillary first molars in a group of 45 volunteers. The volunteers were treated with 10% carbamide peroxide gel with carbopol as the thickening agent (group 1), 2% carbopol gel (group 2), 10% carbamide peroxide paste with poloxamer as the thickening agent (group 3), poloxamer (group 4), or 6.5% hydrogen peroxide strips for 21 days (group 5) (experimental treatment factor). The effects of the experimental treatment were evaluated by microhardness tests performed both before and after treatment (time factor). Data were submitted to split-plot analysis of variance and Tukey tests. Only time showed a statistically significant difference (P .0001). All treatments reduced the enamel microhardness during treatment. Clinically, a reduction in microhardness can be expected after dental bleaching.