Studies in melatonin mechanism of action have suggested that one of them could be the binding of the hormone to calmodulin. We assessed calmodulin-melatonin binding by combining liposome incorporation of calmodulin with separation of free and bound 3H-Melatonin by a rapid ultrafiltration method. Specific binding to calmodulin was saturable, reversible, Ca(++)-dependent, ligand selective, and showed high affinity. Saturation as well as association-dissociation studies revealed that 3H-Melatonin binds to a single site on the calmodulin molecule with a Kd of 188 pM and a total binding capacity Bmax of 35 pM/ug of calmodulin. Displacement experiments showed that the relative order of potency of some compounds for inhibition of 3H-Melatonin was as follows: Melatonin > 6-chloromelatonin > 6-hydroxymelatonin > luzindole > trifluoperazine. The results explain our previously reported melatonin effects such as cytoskeletal rearrangements, inhibition of calmodulin dependent phosphodiesterase activity as well as the modification of Ca(++)-calmodulin electrophoretic mobility. The high affinity of melatonin binding to calmodulin suggests that the hormone is able to modulate cell activity by intracellularly binding to calmodulin at physiologically ranges. Melatonin-calmodulin binding could modulate many intracellular Ca++ functions and thus, the set-point for cell activity will follow the rhythmic circulating levels of the pineal hormone. Moreover, since calmodulin and melatonin are phylogenetically well preserved compounds, their interaction may represent a primary mechanism for both the regulation and the synchronization of cell physiology.