Cyclic nucleotide derivatives have been used as a tool to investigate the existence of distinctive activating and hydrolytic sites on the phosphodiesterase from rat liver activated by cGMP (guanosine 3',5'-monophosphate). This positively cooperative enzyme was stimulated up to 30-fold by 3 microM cGMP when 3 microM cAMP (adenosine 3',5'-monophosphate) was used as substrate. All analogues were less potent activators than cGMP. Most cAMP derivatives were inactive, with two exceptions: 7-deazaadenosine 3',5'-monophosphate and 3'-amino-3'-deoxy-adenosine 3',5'-monophosphate. Benzimidazole ribonucleoside 3',5'-monophosphate, where the two atoms of nitrogen of the pyrimidine ring are missing was a better stimulator than the intact purine-related cyclic derivative. When cAMP and cGMP with identical chemical ligands substituted at the same position were compared, the cGMP analogue was always the more potent activator suggesting that the activating site is sensitive to a guanine-type cyclic nucleotide structure. Degradation of the derivatives by the enzyme was measured by high-performance liquid chromatography: no relation could be established between hydrolysis and effectiveness of activation. In addition, there was no parallelism between inhibitory and activating potency for ten cyclic nucleotide derivatives. Since the chemical interactions between the analogues at the activating site on the one hand and at the catalytic site on the other, are different, it is proposed that the sites are distinct. Consequently, it is suggested that the enzyme operates in steps. In the first activating step, cGMP is fixed by at least two hydrogen bonds at a specific binding site of the enzyme. This is followed by a conformational change of the protein and subsequently a change of the kinetic parameters. In a rather unspecific process and in a second hydrolytic step, several purine-related cyclic nucleotides are converted to the corresponding 5' nucleotides.