Circulating corticosterone levels show an ultradian rhythm resulting from the pulsatile release of glucocorticoid hormone by the adrenal cortex. Since the pattern of hormone availability to corticosteroid receptors is of functional significance, it is important to determine whether there is also a pulsatile pattern of corticosterone concentration within target tissues such as the brain. Furthermore, it is unclear whether measurements of plasma corticosterone levels accurately reflect corticosterone levels in the brain. Given that the hippocampus is a principal site of glucocorticoid action, we investigated in male rats hippocampal extracellular corticosterone concentrations under baseline and stress conditions using rapid-sampling in vivo microdialysis. We found that hippocampal extracellular corticosterone concentrations show a distinct circadian and ultradian rhythm. The PULSAR algorithm revealed that the pulse frequency of hippocampal corticosterone is 1.03±0.07 pulses/hour between 09:00–15:00h and is significantly higher between 15:00–21:00h (1.31±0.05). The hippocampal corticosterone response to stress is stressor-dependent but resumes a normal ultradian pattern rapidly after the termination of the stress response. Similar observations were made in the caudate putamen. Importantly, simultaneous measurements of plasma and hippocampal glucocorticoid levels showed that under stress conditions corticosterone in the brain peaks 20 min later than in plasma but clears concurrently, resulting in a smaller exposure of the brain to stress-induced hormone than would be predicted by plasma hormone concentrations. These data are the first to demonstrate that the ultradian rhythm of corticosterone is maintained over the blood-brain barrier and that tissue responses cannot be reliably predicted from the measurement of plasma corticosterone levels.