In this study we describe the molecular identification, kinetic characterization and biochemical properties of an E-NTPDase and an 5′-nucleotidase in Walker 256 cells. For the ATP, ADP and AMP hydrolysis there were optimum pH in the range 6.5–8.0, and absolute requirement for divalent cations (Mg2+ > Ca2+). A significant inhibition of ATP and ADP hydrolysis was observed in the presence of high concentrations of sodium azide and 0.5 mM of Gadolinium chloride. These activities were insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors. The Km values were 464.2 ± 86.6 μM (mean ± SEM, n = 4), 137.0 ± 31 μM (mean ± SEM, n = 5) and 44.8 ± 10.2 μM (mean ± SEM, n = 4), and Vmax values were 655.0 ± 94.6 (mean ± SEM, n = 4), 236.3 ± 27.2 (mean ± SEM, n = 5) and 177.6 ± 13.8 (mean ± SEM, n = 5) nmol of inorganic phosphate min−1 mg of protein−1 for ATP, ADP and AMP, respectively. Using RT-PCR analysis we identified the mRNA of two members of the ecto-nucleoside triphosphate diphosphohydrolase family (NTPDase 2 and 5) and a 5′-nucleotidase. The presence of NTPDases and 5′-nucleotidase enzymes in Walker 256 tumor cells may be important to regulate the ratio adenine nucleotides/adenine nucleoside extracellularly, therefore motivating tumor growth.