Serotonin (5-HT) has been implicated in a variety of behavioral and physiological processes mediated by the central nervous system. However, the exact nature of 5-HT release under naturalistic or physiologic conditions remains unclear. The present study investigated this issue by employing in vivo microdialysis to examine 5-HT release in the hippocampus, corpus striatum, amygdala, and prefrontal cortex of the rat during manipulations that induced varying behavioral and physiological responses. In each rat, two sites were examined simultaneously during two of the following manipulations: tail pinch, tail pinch with food present, feeding, cat exposure, floating, and swimming. Results indicate that 5-HT levels increased 20-65% above baseline levels in response to each manipulation in all forebrain areas examined. The only significant difference found across manipulations was that tail pinch with food present produced a greater increase in 5-HT than cat exposure, which may be attributable to differences in the degree of general activation induced by these manipulations rather than to specific physiological or behavioral aspects. Furthermore, there was a strong positive correlation between 5-HT release and time spent in alert waking. Finally, there was an overall significantly smaller increase in 5-HT release in the corpus striatum compared to the other three sites. These data suggest that the release of 5-HT in the forebrain is closely related to behavioral state, not to specific behaviors, and that the magnitude of 5-HT release may reflect the degree of activation. In addition, this study suggests there can be some degree of differential pattern of release of 5-HT in the forebrain.