Glucocorticoid hormones are released in rapid hourly hormone bursts by the adrenal gland. These ultradian oscillations are fundamental to hypothalamic-pituitary-adrenal activity and transcriptional regulation of glucocorticoid responsive genes. The physiological relevance of glucocorticoid pulsatility is however unknown. Using a novel automated infusion system, we artificially created different patterns (modulating pulse amplitude) of corticosterone (cort). Identical amounts of cort either in constant or in hourly pulses were infused into adrenalectomized rats. At the end of the infusion period, either during rising or falling concentrations of a cort pulse, animals were exposed to 99 dB noise stress (10 min). Pulsatile cort infusion led to a differential stress response, dependent on the phase of the pulse during which the stress was applied. Although constant administration of cort resulted in a blunted ACTH response to the stressor, a brisker response occurred during the rising phase of plasma cort than during the falling phase. This phase-dependent effect was also seen in the behavioral response to the stressor, which was again greater during the rising phase of each cort pulse. Within the brain itself, we found differential C-fos activation responses to noise stress in the pituitary, paraventricular nucleus, amygdala, and hippocampus. This effect was both glucocorticoid pulse amplitude and phase dependent, suggesting that different stress circuits are differentially responsive to the pattern of glucocorticoid exposure. Our data suggest that the oscillatory changes in plasma glucocorticoid levels are critical for the maintenance of normal physiological reactivity to a stressor and in addition modulate emotionality and exploratory behavior.