Neurons tightly regulate the level of free intracellular calcium in the nanomolar range. Alteration of intracellular calcium (Ca2+i) homeostasis can result in modulation of neuronal excitability and, in extreme cases, neuronal cell death. We hypothesized that bilirubin toxicity-induced neuronal injury is due to an alteration of Ca2+i homeostasis, and that Ca2+i would increase after bilirubin exposure. We utilized the Meridian ACAS confocal interactive laser cytometer system and exposed cultured hippocampal pyramidal neurons (n=16 neurons in 3 experiments) to bilirubin 32 μM bound in a 1:1 molar ratio with human albumin 32 μM at physiological pH. Baseline Ca2+i levels of approximately 200 nM were observed prior to bilirubin toxicity exposure. Immediately following addition of bilirubin solution, Ca2+i increased to a peak level of 993±26 nM at 1 min., then decreased to a new, elevated level of 347±8 nM at 20 min., and remained elevated for up to 45 mins, as long as bilirubin was present in the media. To further determine whether the bilirubin toxicity-induced increase in Ca2+i could modulate neuronal excitability, we tested the neuronal response to the excitatory neurotransmitter glutamate. Sham-treated primary neuronal cultures responded to 50 μM glutamate with an immediate increase in Ca2+i that returned to baseline levels within 6-8 min. after removal of glutamate. In neurons exposed to bilirubin, the initial increase in Ca2+i was observed after glutamate, but neurons were unable to restore resting Ca2+i for up to 40 min. After the removal of glutamate. Thus, in primary hippocampal neurons in culture, bilirubin exposure results in an elevation of Ca2+i. In addition, bilirubin exposure results in increased sensitivity to glutamate exposure because of an inability of bilirubin-exposed neurons to restore resting Ca2+i levels following transient glutamate exposure. The data support the hypothesis that prolonged exposure of neurons to bilirubin results in increased Ca2+i and increased neuronal excitability. The data also provide a cellular mechanism whereby bilirubin exposure can result in delayed neuronal cell death.