Snake venom phosphodiesterase is inactivated by nucleotide analogue 5`-fluorosulfonylbenzoyladenosine. Exonucleolytic activity of the enzyme toward bis-p-nitrophenylphosphate is inhibited with a time-dependent and irreversible manner following pseudo-first-order kinetics. The apparent K₁ and kinact were determined to be 0.16 mM and 0.32 min^(-1), respectively. At the same time, the endonucleolytic function toward plasmid DNA, which was analyzed by agarose gel electrophoresis and by fluorometric method using ethidium bromide, was also inactivated. Kinetic data suggest one mole of the affinity labeling nucleotide was bound per mol of the inactivated enzyme. It appears that the catalytic site responsible for both of the exonuclease and endonuclease activities is located in a single functional domain of the monomeric enzyme molecule.