Trypsin-treated Ca²⁺/calmodulin-dependent phosphodiesterase (Ca²⁺-PDE), which had lost its sensitivity to Ca²⁺-calmodulin, was inhibited by various calmodulin antagonists, trifluoperazine, chlorpromazine, N- (6-aminohexyl) -5-chloro-1-naphth-alenesulfonamide (W-7) and aminoalkyl chain analogues of W-7 (A-3, A-4, A-5, I-240, A-6, A-7). These inhibitory effects were less than those on calmodulin-activated Ca²⁺-PDE. The ability of these compounds to inhibit trypsin-treated Ca²⁺-PDE correlated well with the inhibitory effect on calmodulin-activated Ca²⁺-PDE. W-7 inhibited trypsin-treated Ca²⁺-PDE in a competitive fashion with respect to cyclic GMP and the K₁, value was 300 μM. The inhibition of trypsin-treated Ca²⁺-PDE by W-7 (300 μM) or A-7 (100 μM) was overcome by the addition of excess calmodulin. Trypsin-treated Ca²-PDE can bind to W-7-coupled cyanogen bromide-activated Sepharose 4B in the presence of 1mM EGTA. These results suggest that Ca²⁺-PDE possesses a binding site for calmodulin antagonists and that the binding site for these antagonists on this enzyme may be structurally similar to the binding site on calmodulin itself.