Limited data are available to assess the safety of high levels of hydrogen peroxide in overnight tooth-whitening formulas. The purpose of this study was to assess the effects of hydrogen peroxide on enamel microhardness, pulp penetration, and enamel morphology. Colgate Platinum Professional Overnight Whitening System (Colgate Oral Pharmaceuticals, Inc., Canton, MA, USA) (10% carbamide peroxide, equivalent to 3.5% hydrogen peroxide) was compared with two prototype formulations containing either 7.0% or 12.0% hydrogen peroxide. In the pulp chamber studies, human extracted teeth were exposed to 3.5%, 7.0%, or 12.0% hydrogen peroxide for 30 minutes, 4 hours, or 7 hours. Microhardness, electron spectroscopy for chemical analysis, and atomic force microscopy evaluations were made from enamel blocks cut from human extracted molars. The enamel blocks were evaluated following 14 7-hour treatments (98 h total). At 7 hours' post-treatment, hydrogen peroxide penetrated the pulp chamber at 23.12 +/- 10.09, 24.58 +/- 6.90, and 26.39 +/- 5.43 microg for 3.5%, 7.0%, and 12.0% hydrogen peroxide, respectively. With regard to enamel morphology, pulp penetration, microhardness, and elemental composition, no statistically significant differences were observed between treatment groups following 98 hours of treatment. Hydrogen peroxide does not adversely affect enamel morphology or microhardness. The levels recovered in pulp indicate that hydrogen peroxide is not expected to inhibit pulpal enzymes. Overnight tray products containing levels of hydrogen peroxide of 3.5%, 7.0%, and 12.0% are not expected to adversely affect the enamel or pulpal enzymes. Additional safety studies are needed to assess the potential for tooth sensitivity and gingival irritation.