This in situ study evaluated the microhardness of sound and demineralized enamel and dentin submitted to treatment with 10% carbamide peroxide for three weeks. A 10% carbamide peroxide bleaching agent--Opalescence/Ultradent (OPA)--was evaluated against a placebo agent (PLA). Two hundred and forty dental fragments--60 sound enamel fragments (SE), 60 demineralized enamel fragments (DE), 60 sound dentin fragments (SD) and 60 demineralized dentin fragments (DD)--were randomly fixed on the vestibular surface of the first superior molars and second superior premolars of 30 volunteers. The volunteers were divided into two groups that received bleaching or the placebo agent at different sequences and periods at a double blind 2 x 2 crossover study with a wash-out period of two weeks. Microhardness tests were performed on the enamel and dentin surface. The SE and DE submitted to treatment with OPA showed lower microhardness values than the SE and DE submitted to treatment with PLA. There were no statistical differences in microhardness values for SD and DD submitted to the treatment with OPA and PLA. The results suggest that treatment with 10% carbamide peroxide bleaching material for three weeks alters the enamel microhardness, although it does not seem to alter the dentin microhardness.