Plasma corticosterone levels in rodents are characterised by a diurnal rhythm with low levels in the morning and markedly higher levels towards the evening, the activity phase of these nocturnal animals. Apart from diurnal changes, plasma corticosterone levels also display an ultradian rhythm [1], possibly resulting from its pulsatile secretion by the adrenal cortex [2]. At present it is unknown whether this ultradian rhythm in glucocorticoid hormone persists across the blood-brain barrier and can be found in the extracellular space of the brain. To clarify this issue, we designed a series of in vivo microdialysis studies to measure corticosterone levels in the rat hippocampus, a principal site of glucocorticoid action in the brain. Importantly, because the extracellular fluid is devoid of corticosterone binding proteins, dialysate levels represent the free, i.e. the biologically active fraction, of this glucocorticoid [3]. Male and female Wistar rats were equipped with a guide cannula under isoflurane anaesthesia. Seven days later a microdialysis probe was inserted, under isoflurane anaesthesia, into the hippocampus. After 2 days, collection of dialysate samples was started at 05:00 h and continued for 48 h. Sample intervals were 10 min or 30 min between 08:00-22:00 h and 22:00-08:00 h, respectively. On the second day animals were subjected to novelty (30 min) or swim stress (15 min, 25°C), or left undisturbed in their home cage. A sensitive radioimmunoassay was used to measure dialysate corticosterone. Data were analysed using Pulsar PC and SPSS software. It was found that hippocampal free corticosterone levels show a clear pulsatile pattern in both male and female rats. Interestingly, in both sexes the pulse amplitude increased towards the evening. A comparison between male (n=15) and female (n=10) rats over a 12 h period (09:00-21:00 h), revealed no significant gender differences (P > 0.05, Student’s t test) in the total number of pulses (male (m): 14.6±0.5 vs. female (f): 14.5±1.2 pulses) and the pulse amplitude (0.11±0.01 (m) vs. 0.09±0.01 (f) µg/dl). Moreover, the mean corticosterone level was similar in male and female rats (0.14±0.02 (m) vs. 0.11±0.01 (f) µg/dl). While both novelty and swim stress caused a rise in free corticosterone in male and female rats, the ultradian rhythm was rapidly restored after termination of the stressors (maximal effect in male rats: novelty: 0.27±0.08 µg/dl, swim: 0.79±0.34 µg/dl; female rats: novelty 0.27±0.05 µg/dl, swim: 1.27±0.33 µg/dl). These data demonstrate for the first time the existence of an ultradian rhythm of free corticosterone in the hippocampus of rats. Such an ultradian pattern of hormone levels could be of great significance for glucocorticoid action in the hippocampus during ongoing conditions and for the role of this brain structure in the regulation of the hypothalamic-pituitary-adrenal axis and other glucocorticoid-sensitive systems over the diurnal cycle.