Calixarenes modified with two or three Zn(II)-2,6-bis(aminomethyl)pyridyl groups, 3-[Zn]2 and 5-[Zn]3, respectively, were investigated as models for dinuclear and trinuclear metallo-enzymes that catalyze the cleavage of phosphate diesters. Under neutral conditions, 0.48 mM of 3-[Zn]2 causes a rate acceleration of 23 000 in the transesterification of the RNA model substrate 2-hydroxyproyl-p-nitrophenyl phosphate (HPNP, 0.19 mM). Comparison with the activities of a mononuclear complex 2-[Zn] and a reference complex lacking the calixarene backbone 1-[Zn] shows that the catalysis is due to cooperative action of the Zn(II) centers and indicates that hydrophobic effects contribute to the catalysis. Saturation kinetics and pH variation studies demonstrate that the high catalytic activity of the flexible complex 3-[Zn]2 originates from a very high substrate binding affinity, affording a Michaelis-Menten complex in which the substrate is converted with a relatively moderate rate. A rigid analogue 4-[Zn]2 exhibits both a lower substrate binding strength and a lower catalytic rate. This demonstrates the importance of a certain flexibility between the cooperating catalytic centers. The trinuclear complex 5-[Zn]3 induces a rate acceleration of 32 000 times, and shows a decreased substrate binding and an increased catalytic rate compared to its dinuclear analogue 3-[Zn]2. In a possible mechanism two Zn(II) ions activate the phosphoryl group and another activates the B-hydroxyl group of HPNP.