Purpose: To measure the demineralization capacity of 37% phosphoric acid on surface and subsurface bovine enamel after bleaching with hydrogen peroxide (H2O2). Materials and Methods: Three equally-sized sections with 16 mm2 of exposed enamel surface were obtained from the enamel of 10 bovine incisors. One specimen sample from each crown was assigned to one of three groups (n = 10): group I, no bleaching agent; group II, bleached with 38% H202 for 20 min; or group III, 30% H202 for 60 min. After 24 h, the thickness of specimens was measured and they were immersed in 37% phosphoric acid solution, of which 5-ml aliquots were collected at 30 s and 60 s. Specimens were then ground to a depth of 25 µm and again immersed in 37% phosphoric acid solution. This procedure was repeated for enamel depths of 50 and 100 µm. Ca2+ concentrations in the phosphoric acid aliquots were measured by atomic absorption spectroscopy. Results: No significant differences were found in the total amounts of extracted Ca2+ between bleached and unbleached specimens (F = 0.142; p = 0.869). The amount of Ca2+ extracted was similar among the four depth levels in the unbleached and in the 30% H202 bleached specimens. A significantly larger amount of Ca2+ was obtained at 25 µm depth (subsurface) from specimens treated with 38% H202. Conclusions: Pre-bleaching with 38% H202 significantly increased the decalcifying effect of phosphoric acid on subsurface enamel at a depth of 25 µm compared to 100 µm, whereas pre-bleaching with 30% H202 did not modify this effect at any level. Keywords: hydrogen peroxide, decalcifying, subsurface enamel, phosphoric acid, enamel acid etching, atomic absorption spectroscopy