Excellent progress has been made toward understanding the physiology and pharmacology of specific calcium-related cellular processes of the brain, but few studies have provided an integrated view of brain calcium kinetics. To further the knowledge of the size and binding properties of brain calcium compartments, the authors have conducted a series of experiments in hippocampal brain slices exposed to high and low extracellular calcium. Slices were incubated in buffers containing 0.001 to 4.5 mmol/L calcium for up to 75 minutes. Slice calcium content was analyzed by three methods: exchange equilibrium with 45Ca, synchrotron-radiation-induced x-ray emission, and inductively coupled plasma. Data were analyzed using a model based on a Langmuir isotherm for two independent sites, with additional extracellular and bound compartments. In parallel experiments, altered low calcium had no effect on slice histology and only mild effects on slice adenylates. When combined with prior 45Ca and fluorescent probe binding experiments, these results suggest that there are at least five kinetically distinct calcium compartments: (1) free extracellular (approximately 10%); (2) loosely associated extracellular plasma membrane (approximately 55%); (3) intracellular compartment with moderate avidity (approximately 17%); (4) tightly bound, nonexchangeable intracellular compartment ( approximately 15%); and (5) free cytoplasmic (<0.01%). If only the third compartment is considered a potential calcium buffer, then the buffering ratio is calculated to be approximately 2,700:1, but if the second compartment is also included, then the buffering ratio would be approximately 13,000:1. This may explain the wide range of estimates observed by fluorescent probe studies.