INTRODUCTION: The pineal gland hormone, melatonin, has been shown by numerous studies to inhibit the proliferation of estrogen receptor α (ERα)-positive breast cancer cell lines. Here, we investigated the role of melatonin in the regulation of breast cancer cell invasion. METHODS: Three invasive MCF-7 breast cancer cell clones - MCF-7/6, MCF-7/Her2.1, and MCF-7/CXCR4 cells - were employed in these studies. All three cell lines exhibited elevated phosphorylation of the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) as determined by Western blot analysis. The effect of melatonin on the invasive potential of these human breast cancer cells was examined by matrigel invasion chamber assays. The expression and proteinase activity of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were analyzed by Western blot analysis and gelatin zymography, respectively. RESULTS: Melatonin (10-9 M) significantly suppressed the invasive potential of MCF-7/6 and MCF-7/Her2.1 cells as measured by matrigel invasion chamber assays, and significantly repressed the proteinase activity of MMP-2 and MMP-9. In MCF-7/CXCR4 cells, melatonin significantly inhibited stromal-derived factor-1 (SDF-1/CXCL12) induced cell invasion and activity of MMP-9. Elevated expression of the MT1 melatonin receptor further enhanced, while luzindole, an MT1/MT2 antagonist, abrogated melatonin's anti-invasive effect, suggesting that melatonin's effect on invasion is mediated, principally, through the MT1 receptor. Furthermore, melatonin repressed the phosphorylation of p38 MAPK in MCF-7/Her2.1 cells and blocked stromal-derived factor-1 (SDF-1) induced p38 phosphorylation in MCF-7/CXCR4 cells. SB230580, a p38 inhibitor, was able to mimic, while transfection of the cells with a constitutively-active MKK6b construct blocked melatonin's effect on cell invasion, suggesting that the anti-invasive action of melatonin is mediated through the p38 pathway. CONCLUSIONS: Melatonin exerts an inhibitory effect on breast cancer cell invasion through down-regulation of the p38 pathway, and inhibition of MMP-2 and MMP-9 expression and activity.