Calmodulin-dependent cyclic nucleotide phosphodiesterase (EC 3.1.4.17) was purified from silkworm pupal fat body by DEAE-Sepharose and calmodulin-Sepharose column chromatographies. The calmodulin-dependent activity accounted for 3–5% of the total cytosol phosphodiesterase activity, and was purified approx. 5000-fold from the fat body extract. The purified phosphodiesterase readily lost sensitivity to calmodulin during storage at 4°C or limited digestion by trypsin. The enzyme could hydrolyze both cyclic AMP and cyclic GMP, and was activated 2- to 4-fold by calmodulin and Ca 2+. The molecular weight of the enzyme was calculated from the sedimentation constant and Stokes radius to be 115,000. Kinetic analyses suggested the enzyme molecule had at least two distinct sites for the cyclic nucleotides.