The effects of forced swimming for 30 min on extracellular 5-hydroxytryptamine (5-HT) levels were examined in five brain regions in rats using in vivo microdialysis. A single dialysis probe was implanted under surgical anesthesia into either the striatum, ventral hippocampus, frontal cortex, amygdala, or lateral septum on the day before the study. Dialysate content of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) was measured by HPLC. Forced swimming elevated extracellular levels of 5-HT in the striatum to a maximum of 90% above baseline. In contrast, forced swimming reduced 5-HT levels in the amygdala and lateral septum to 50 and 40% of baseline, respectively. In the hippocampus and frontal cortex, 5-HT levels were not altered significantly by forced swimming. In all five brain regions, forced swimming reduced 5-HIAA levels to 45–60% of baseline. These results suggest that forced swimming modulates 5-HT neurotransmission in a regionally specific manner. Aside from being a significant biological stressor, the forced swimming test is used as an animal behavioral model to detect antidepressant drugs, including drugs that alter 5-HT neurotransmission. It is possible that the alterations of extracellular levels of 5-HT produced by forced swimming in certain brain regions may be associated with the ability of antidepressant drugs to selectively alter behavioral performance during the forced swimming test.