In the cardiovascular system extracellular ATP, ADP, AMP and adenosine play an important role in thrombosis and inflammation, modulating processes how platelet aggregation, endothelial cell activation, vasodilatation and vasoconstriction. Furthermore, in the heart, they are involved in contractile, chronotropic and arrhythmic effects. Members of several enzyme families are capable of hydrolyzing extracellular adenine nucleotides, modulating the purine signaling. These include the family of E-NTPDase and 5'-nucleotidase. The objective of the present study is to characterize the enzymes involved in ATP, ADP e AMP hydrolysis in cardiac microsomes of adult male rats. The microsomal fraction was obtained from differential centrifugation of ventricles homogenate. The preparation was preincubated for 10 minutes at 37°C and the reaction was started by the addition of ATP, ADP or AMP to a final concentration of 2.0 mM. The reaction was stopped by the addition of 10% trichloroacetic acid (TCA, 5% final concentration) and the amount of Pi liberated was measured by the method of Chan et al., 1986. The protein concentration and the incubation time were chosen in order to ensure the linearity of the reaction. Protein was measured according to Bradford, 1976. Then, for ATP assay were used 20 mg/tube of protein and 2 minutes of incubation. For ADP and AMP assays were used 50mg/tube of protein and 10 minutes of incubation. The mitochondria ATPase inhibitor (olygomycin 2mg/mL) was used in all enzymatic ATP and ADP assays and the adenylate kinase inhibitor, NEM (4.0 mM), was used in all experiments of ADP hydrolysis. Both enzymes (NTPDase plus 5´-nucleotidase) were cation-dependent and showed a preference for ion Mg²⁺. In addition, both activities were blockade by metal ion chelator (EDTA). Our results demonstrate ATP, ADP and AMP hydrolysis by heart microsomes obtained from rats. The study of the metabolism of these nucleotides modulating the ratio nucleotides/nucleoside in the heart could be important for the understanding of the processes related to signaling of these molecules such as described above.