The aim of the present work was to study whether physiological doses of melatonin (1nM) modified DNA synthesis in MCF-7 human breast cancer cells. Exponentially growing MCF-7 cells were incubated for 24 h with thymidine (2mM) for blocking mitosis and synchronizing the cell division cycle. Synchronization was assessed by a flow cytometry study which showed that after release from excess thymidine, 82.3% of the cells were in phase G1. Lots of these synchronized cells were pulsed for 1h with [3H]deoxythymidine ([3H]dThy) or [3H]dThy + melatonin, at 0,3,6,9,12,15 or 24 h from the release of the mitotic arrest. The exposition of these synchronized MCF-7 cells to melatonin for only 1h, significantly inhibited [3H]dThy incorporation when it was at 6 or 9 h. after release from mitotic block, at a time when DNA precursor incorporation was the highest and the number of cells in S phase was maximum. We conclude that, at least in part, melatonin antiproliferative effects on MCF-7 cells could be mediated by a reduction of DNA synthesis.