This study determined whether muscle contraction causes an increase in extracellular levels of serotonin (5-HT) in the rostral (rVLM) or caudal ventrolateral medulla (cVLM) in anesthetized rats. Muscle contraction, evoked by tibial nerve stimulation, increased mean arterial blood pressure (MAP) by 27 ± 4 mmHg ( n = 8). In addition, 5-HT levels in the rVLM were elevated by 65 ± 9% during the contraction ( n = 8). Results were similar over two repeated contractions. In contrast, muscle contraction increased MAP, but not 5-HT, levels in the cVLM ( n = 6). Tibial nerve stimulation after muscle paralysis had no effect on either MAP or 5-HT levels in both rVLM and cVLM. Microdialysis of a 5-HT_1A agonist, 8-OH-DPAT (10 mM), into the rVLM for 30 min ( n = 6) blunted the MAP change and reduced 5-HT release during contraction. Administration of NAN-190, a 5-HT_1A antagonist, into the rVLM had no effect on 5-HT release and cardiovascular responses during muscle contraction and blocked the changes in 5-HT, MAP, and heart rate to static contraction after subsequent microdialysis of 8-OH-DPAT. Results demonstrate that 5-HT levels in the rVLM increase during muscle contraction and that 5-HT_1A-receptor activation in the rVLM blunts MAP response to muscle contraction via a decrease in the extracellular concentration of 5-HT.