The pineal hormone, melatonin, has been shown to inhibit the proliferation of the estrogen receptor alpha (ERα)-positive macrophage chemotactic factor (MCF)-7 human breast cancer cells. Previous studies from other systems indicate that melatonin modulates the calcium (Ca2+)/calmodulin (CaM) signaling pathway either by changing intracellular calcium concentration ([Ca2+]i) via activation of its G-protein coupled membrane receptors, or through a direct interaction with CaM. In this study, although melatonin alone had no effect on basal [Ca2+]i in MCF-7 cells, it significantly enhanced the elevation of [Ca2+]i induction by extracellular adenosine triphosphate (ATP), which increases [Ca2+]i via the G protein-coupled P2y-purinoceptor and the phospholipase C (PLC) pathway. Pretreatment of MCF-7 cells with 10–7 M melatonin increased the 10–5 M ATP-induced [Ca2+]i peak change from 79.4 ± 11.6 nM to 146.2 ± 22.3 nM. Furthermore, without changing total cellular CaM levels, melatonin markedly increased the amount of membrane-bound CaM to 237 and 162% of control levels after 1 and 6 hr of treatment, respectively. Cytosolic CaM levels were also elevated to 172% of control after 6 hr of melatonin treatment. Correlative growth studies demonstrated that ATP (10−5 M) can stimulate MCF-7 cell growth, that melatonin can suppress MCF-7 cell proliferation, but that pretreatment of MCF-7 cells with melatonin followed by ATP(10–5 M), like 10–4 M ATP can further suppress MCF-7 cell proliferation; this indicates that melatonin's potentiation of ATP induced [Ca2+]i may be above the threshold for cell growth. Given the important role of [Ca2+]i and CaM in tumor cell homeostasis and proliferation and melatonin's modulation of [Ca2+]i, melatonin's effects on the Ca2+/CaM signaling pathway may play an important role in mediating the growth-inhibitory effect of melatonin on MCF-7 human breast cancer cells.