Central serotonin [5-hydroxytryptamine (5-HT)] is involved in the aetiology of numerous disease states, including depression and anxiety disorders. Studies have shown that exposure of rats to animal tests of anxiety increases extracellular 5-HT in the cortex or hippocampus determined by in vivo microdialysis. To discriminate whether this increase is caused by the aversive conditions of an animal test for anxiety or by an unconditioned stressor evoking mainly arousal, the present study investigates the effects of an unconditioned acoustic stimulus and exposure to the elevated plus maze (X-maze), respectively, on the release of 5-HT in the ventral hippocampus compared with hippocampal 5-HT release in the home cage and in a non-aversive unfamiliar environment in freely moving rats. Our results showed a distinct pattern of 5-HT release in the ventral hippocampus depending on the stimulus used. Exposure to the X-maze for 20 min was accompanied by an ‘anxious’ behaviour in the rats and increased extracellular 5-HT to 165% of basal release, whereas exposure to a less aversive ‘deactivated’ plus maze (115 ± 6%) or to white noise for 20 min in the familiar surroundings of the home cage (98 ± 6%) did not change hippocampal 5-HT release significantly, despite similar behavioural activation indicated by increased locomotor activity. While both the X-maze and white noise may model anxiety and stress to a certain extent, it seems that the X-maze is more aversive. The results suggest a close relationship between anxiety-related behaviour, but not arousal/non-specific behavioural activation, and 5-HT release in the ventral hippocampus.