In this study, we have identified the E-NTPDase family members and ecto-5'-nucleotidase/CD73 in rat heart left ventricle. Moreover, we characterize the biochemical properties and enzyme activities from synaptosomes of the nerve terminal endings of heart left ventricle. We observe divalent cation-dependent enzymes that presented optimum pH of 8.0 for ATP and ADP hydrolysis, and 9.5 for AMP hydrolysis. The apparent K(M) values are 40 microM, 90 microM and 39 microM and apparent V(max) values are 537, 219 and 111 nmol Pi released/min/mg of protein for ATP, ADP and AMP hydrolysis, respectively. Ouabain, orthovanadate, NEM, lanthanum and levamisole do not affect ATP and ADP hydrolysis in rat cardiac synaptosomes. Oligomycin (2 microg/mL) and sodium azide (0.1 mM), both mitochondrial ATPase inhibitors, inhibit only the ATP hydrolysis. High concentrations of sodium azide and gadolinium chloride show an inhibition on both, ATP and ADP hydrolysis. Suramin inhibit more strongly ATP hydrolysis than ADP hydrolysis whereas Evans blue almost abolish both hydrolysis. AMP hydrolysis is not affected by levamisole and tetramisole, whereas 0.1 mM ammonium molybdate practically abolish the ecto-5'-nucleotidase activity. RT-PCR analysis from left ventricle tissue demonstrate different levels of expression of Entpd1 (Cd39), Entpd2 (Cd39L1), Entpd3 (Cd39L3), Entpd5 (Cd39L4) Entpd6, (Cd39L2) and 5'-NT/CD73. By quantitative real-time PCR we identify the Entpd2 as the enzyme with the highest expression in rat left ventricle. Our results contribute to the understanding about the control of the extracellular nucleotide levels in and cardiac system.