Peroxidase are unspecific enzymes and several molecules can be oxidized by the active forms HRP-I and HRP-II in the classic cycle of peroxidase, which depends on hydrogen peroxide or organic hydroperoxides. On the other hand, only NADH, dihydroxyfumarate and the auxin indole-3-acetic (IAA) have been described as substrate for HRP in a reaction independent of hydrogen peroxide. The objective of this work is to study the mechanism for which b-dicarbonyl compounds are oxidized by horseradish peroxidase (HRP). We verify through studies of oxygen uptake , that the acetylacetone (3 mM) also can be oxidized by the HRP (0,5 mM) in the peroxide absence, in phosphate buffer pH 7,4, consuming 50% of the oxygen present in solution in 5 minutes of reaction, compared to less than 10% of others b-dicarbonyls compounds (dimedon and acetoacetate), in the same conditions. It was also observed, in the absorption spectrum during the reaction course with the acetylacetone, that the native enzyme is transformed in HRP-III; having still produced reactive species of oxygen (EROs), that had been detected by luminol-dependent chemiluminescence. The integral of light emission in the reaction of acetylacetone (1 mM) with HRP (0,01 mM), using luminol ( 0,1 mM), in phosphate buffer pH 7,4, was about 150 times superior in relation the dimedon and the acetoacetate, in the same conditions of reaction. In summary, it was observed that acetylacetone behaves as a substrate for HRP in reaction hydrogen peroxide-independent and produces EROs.