Modulation of intracellular free calcium levels is the primary second messenger system of the neuronal glutamatergic system, playing a role in regulation of all major cellular processes. The protein neuregulin (NRG) beta1 acts as an extracellular signaling ligand in neurons, rapidly regulating currents through ionotropic glutamate receptors. The effect NRG may have on glutamate-induced changes in intracellular free calcium concentrations has not been examined, however. In this study, cultured embryonic rat hippocampal neurons were treated with NRGbeta1 to determine a possible effect on glutamate-induced intracellular calcium levels. Long-term (24 h), but not short-term (1 h), incubation with NRGbeta1 resulted in a significantly greater glutamate-mediated acute peak elevation of intracellular calcium levels than occurred in vehicle-treated neurons. Long-term NRGbeta1 incubation significantly enhanced calcium increase induced by specific stimulation of metabotropic glutamate receptors, but did not significantly alter the N-methyl D-aspartate (NMDA)- or KCl-induced calcium increase and paradoxically decreased the effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) treatment on intracellular calcium. Metabotropic glutamate receptors cause increased intracellular free calcium via release of calcium from intracellular stores; thus this system was examined in more detail. NRGbeta1 treatment significantly (greater than 2-fold) enhanced calcium release from endoplasmic reticulum stores after stimulation of ryanodine receptors with caffeine, but did not significantly increase calcium release from endoplasmic reticulum mediated by inositol trisphosphate (IP3) receptors. In addition, ryanodine receptor inhibition with ruthenium red prevented the glutamate-induced increase in intracellular calcium levels in NRGbeta1-treated neurons. These data show that long-term NRGbeta1 treatment can enhance glutamate-induced peak intracellular calcium levels through metabotropic glutamate receptor activation by increasing endoplasmic reticulum calcium release through ryanodine receptors.