A specific antibody combined with a fluorescein-labeled immunoglobulin was used to investigate the topographic distribution of melatonin in a variety of cells of different origins. Positive identification of both nuclear and cytosolic melatonin was confirmed in all the tested cells: Swiss 3T3 mouse fibroblasts, BCG1 bovine granulosa, NB41A3 mouse neuroblastoma, F9 mouse teratocarcinoma, MDCK normal canine kidney derived and human HeLa cell lines, as well as in human peripheral blood mononuclear leukocytes and rat splenic cells. In 3T3 mouse fibroblasts melatonin immunofluorescence partially colocalized with actin and serotonin immunostaining, but not with tubulin or actin stress fibers. Several distinct patterns of subcellular melatonin distribution, different from the bromodeoxyuridine-labeled replication profiles, have been discerned throughout the cell cycle of synchronized 3T3 cells. In addition, synchronized 3T3 mouse fibroblasts cultured in the presence of 10(-3) M melatonin progressed more slowly through the cell cycle than control cells. These results suggest that melatonin may interact directly with nuclear and cytoskeletal structures probably affecting different cell functions such as cell cycle control, subcellular organization, and genome stability.