Abstract 1. 1. A 5′(3′)-nucleotidase, which preferably hydrolyzed 3′-dTMP and 3′-dUMP, was highly purified from rabbit spleen cytosol. 2. 2. The enzyme also hydrolyzes 3′-UMP, 5′-dUMP and guanine nucleotides, but does not hydrolyze any adenine nucleotides or cytosine nucleotides. 3. 3. The activity is dependent upon Mg 2+, Co 2+ or Mn 2+; the addition of deoxyinosine stimulates the activity, and the pH optimum for the hydrolysis of 3′-dTMP is 7.0. 4. 4. Although the catalytic properties of the enzyme are similar to the 5′(3′)-nucleotidase from rat spleen, these nucleotidases differ in their molecular disposition. 5. 5. The charge state of the rabbit enzyme is slightly basic, and the subunit r is about 27kDa, while the value of the rat enzyme is 26 kDa. 6. 6. Immunochemical experiments with the use of antibodies against the purified nucleotidase indicate that enzymes from rabbit spleen and from rat spleen are composed of different polypeptides.