Erythropoietin (Epo) has been shown to afford neuroprotection in many experimental models. Although the cytosolic Ca2+ concentration ([Ca2+)](i)) is an important factor regulating cell survival, the effects of Epo on [Ca2+](i) in neurons are not fully elucidated. We studied the effects of human recombinant Epo on [Ca2+](i) of rat primary cortical neurons in normal and excitotoxic conditions. Changes in [Ca2+](i) were measured using fura-2 microfluorometry in rat primary cortical cultures. In the control condition with 2 mM Mg2+ in the bath solution, Epo at 4 u/ml significantly increased the fluorescence ratio, but the Epo-induced increase in the fluorescence ratio was abolished by omission of Ca2+ from the bath solution and by the addition of cadmium. Omission of Mg2+ supplementation with glycine resulted in basal and periodic increases in the fluorescence ratio, due to sustained activation of N-methyl-D-asparate (NMDA) receptors. Epo at 0.4 and 4 u/ml significantly decreased the fluorescence ratio in this condition, and this effect was attenuated by the phosphoinositide 3-kinase (PI3K) inhibitors, LY 294002 and wortmannin, and the Ca-activated K channel blocker, iberiotoxin. In the presence of Mg2+ and exogenous glutamate, 4 but not 0.4 u/ml Epo slightly but significantly reduced the [Ca2+](i) elevation. These results suggest that Epo increased [Ca2+](i) in cortical neurons by inducing Ca2+ entry in the control condition but decreased [Ca2+](i) in the Mg2+-free excitotoxic condition, at least in part via PI3K-dependent activation of Ca-activated K channels. Reduction of [Ca2+](i) by Epo in the excitotoxic condition may contribute to neuroprotection. (C) 2011 Elsevier B.V. All rights reserved.