A novel 3'-nucleotidase, which preferably hydrolyzed 3'-dTMP and 3'-dUMP, was isolated and purified from rabbit spleen cytosol. Since the main catalytic properties of the enzyme were similar to the rat liver nucleotidase reported by Fritzson and Smith, we also purified the latter enzyme from rat spleen. These two enzymes were different in several points: the substrate restriction, the molecular mass and the apparent charge. Further analyses with the antiserum raised in rat against the 3'-deoxyribonucleotidase evidenced that these enzymes were immunochemically different as well.