Calcium and calcium-dependent systems have been long implicated in the induction of epilepsy. We have previously observed that intracellular calcium ([Ca2+]i) levels remain elevated in cells undergoing epileptogenesis in the hippocampal neuronal culture (HNC) model. In this study, we employed the hippocampal neuronal culture (HNC) model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs) for the life of the neurons in culture to investigate alterations in [Ca2+]i homeostatic mechanisms that may be associated with the 'epileptic' phenotype. [Ca2+]i imaging fluorescence microscopy was performed on control and 'epileptic' neurons with two different fluorescent dyes ranging from high to low affinities for [Ca2+]i. We measured baseline [Ca2+]i levels and the ability to restore resting [Ca2+]i levels after a brief 2-min exposure to the excitatory amino acid glutamate in control neurons and neurons with SREDs. Neurons manifesting SREDs had statistically significantly higher baseline [Ca2+]i levels that persisted for the life of the culture. In addition, the 'epileptic' phenotype was associated with an inability to rapidly restore [Ca2+]i levels to baseline following a glutamate induced [Ca2+]i load. The use of the low affinity dye Fura-FF demonstrated that the difference in restoring baseline [Ca2+]i levels was not due to saturation of the high affinity dye Indo-1, which was utilized for evaluating the [Ca2+]i kinetics at lower [Ca2+]i levels. Peak [Ca2+]i levels in response to glutamate were the same in both 'epileptic' and control neurons. While [Ca2+]i levels recovered in approximately 30 min in control cells, it took more than 90 min to reach baseline levels in cells manifesting SREDs. Alterations of [Ca2+]i homeostatic mechanisms observed with the 'epileptic' phenotype were shown to be independent of the presence of continuous SREDs and persisted for the life of the neurons in culture. Epileptogenesis was shown not to affect the degree or duration of glutamate induced neuronal depolarization in comparing control and 'epileptic' neurons. The results indicate that epileptogenesis in this in vitro model produced long-lasting alterations in [Ca2+]i regulation that may underlie the 'epileptic' phenotype and contribute to the persistent neuroplasticity changes associated with epilepsy.