A heterogeneous class of proteins exhibit within their sequence a particular structure, named EF-hand, able to bind calcium with high affinity. These calcium binding proteins have been described in most cells and tissues and are suggested to work as calcium buffers, thereby participating in the regulation of calcium-dependent cellular activity. Recent circumstantial evidences suggest that calcium binding proteins may serve other functions as well, possibly as enzyme modulators. Since 3',5'-cyclic nucleotide phosphodiesterase is a well-known calmodulin-modulated enzyme, in this work we studied the effect in vitro of different purified calcium binding proteins on the activity of this enzyme. Among the proteins tested, calmodulin and recombinant rat brain parvalbumin could stimulate the 3',5'-cyclic nucleotide phosphodiesterase activity in vitro, whereas rabbit muscle parvalbumin, rat renal and brain calbindin D28K, and bovine brain S-100B were ineffective. Immunoprecipitation with the specific antiserum completely abolished either calmodulin or recombinant brain parvalbumin activation of 3',5'-cyclic nucleotide phosphodiesterase. Moreover, while the presence of calcium in the incubation mixture was critical in the calmodulin-mediated stimulation of the enzyme, it did not modify the effect of the recombinant brain parvalbumin. We suggest that, in addition to calmodulin, parvalbumin may be a regulator of 3',5'-cyclic nucleotide phosphodiesterase, and possibly of other yet to be identified enzymes in certain tissues.