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The activity of a highly purified nucleotidase from rat liver cytosol which splits certain 3'- anti 5'-nucleotides, Was studied in the presence of each of 36 (different nucleic acid nucleic acid constituents including14C, labeled and chemically modified nucleotides. It was found that the compounds either inhibited or had no effect, on dephosphorylation of thymidine 5'-phosphate, which was used as substrate for measuring 5'nucleotidase activity. On the other hand , the 3'-nucleotidase activity, which was measured with uridine 3'-phosphate as substrate, was stimulated 2.6 times by deoxy- guanosine and, furthermore, by dexyguanosine and, furthermore, by deoxyinosine, thymidine, deoxyuridine and inosine in decreasing order of effectiveness. Experiments with various phosphorylated derivatives of thymidine indicated that a 5'-phosphoryl group increase the stimulating effect of the nucleoside whereas a 3'-phosphoryl substitution reduces its ability to activate the enzyme. Di-and triphosphates were less stimulatory than the mono phosphate. The results are interpreted to indicate that the same catalytic site is responsible for the hydrolysis of the 3'-and 5'-nucleotides and that the enzyme possesses a regularly site, topographically different from the catalytic site, at which the deoxyribonucleic acid constituents act as stimulators of enzyme activity. [ABSTRACT FROM AUTHOR]