Purified snake venom phosphodiesterase (M.W. 110 KDa, EC 3.1.4.1) undergoes limited proteolysis by subtilisin treatment. The proteolytic reaction yields an enzymatically active species with M.W. of 100 KDa that can be separated from cleaved peptide mixture by gel filtration. It was investigated to observe whether the selective cleavage of phosphodiesterase molecule elicited by subtilisin treatment would perturb the active site structure associated with bifunctional characteristics of the enzyme. The proteolyzed phosphodiesterase fully retained both exo- and endonuclease functions. However, increased endonucleolytic activity for plasmid DNA as well as the changes in apparent $K_m$ and $V_{max}$ associated with exonuclease activity were observed in the subtilisin-digested phosphodiesterase. Estimated free energy of activation using bis-p-nitrophenylphosphate as a substrate was revealed to be 19.9 cal/mol which is higher than that of native enzyme. Results of thermal inactivation studies and of susceptibility to inhibitors suggest remarkable structural changes arising from the limited proteolysis of the enzyme. It is postulated that a conformational change takes place in the phosphodiesterase molecule by limited subtilisin digestion, while the enzymatic function remains preserved.