Calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase (EC 3.1.4.17) Mr 63,000 and Mr 60,000 from the brain as well as Mr approximately 59,000 species from the heart, have been compared with respect to their steady-state kinetic parameters for the hydrolysis of cAMP, cGMP and their 2'-O-anthraniloyl- and 2'-O-(N-methylanthraniloyl)-derivatives. Kinetic studies with the native substrates indicate high Mr brain enzyme to be cGMP specific whereas low Mr brain and heart enzymes to be nonspecific. In addition, the isozymes studied here appear to be kinetically distinct from those previously isolated form bovine brain tissues. Substitution at 2'-O-position of the cyclic nucleotides gave rise to Vmax values ranging 1-11% of those observed with the native substrates, with minimal effect on Km. The isozymes with exception of heart isoform gave higher Km and Vmax with the anthraniloyl derivatives. This effect is thought to be related to the formation of an intramolecular hydrogen bond which leads to decreased electrostatic interactions between the active-site side chains and the pseudo-substrates.