Prostanoids are known to regulate several physiological functions and to play an important role in certain pathophysiological situations such as inflammation. Prostaglandin E₂ receptors (EP) are members of the G protein-coupled receptor superfamily. Four subtypes have been described: EP2 and EP4 (coupled to G_s proteins) and EP1 and EP3 (coupled to G_i/o proteins). To date, the function of the prostanoid system in the brain has not been well characterized. The locus coeruleus (LC), the main noradrenergic nucleus in the brain, has been described to express the EP3 receptor. The aim of this study was to characterize the functional relevance of EP3 receptors in the LC by single-unit extracellular recordings in rat brain slices. We performed concentration-effect curves for different endogenous derivatives and selective agonists of EP3 receptors. Thus, increasing concentrations of the EP3/EP1 agonist sulprostone (0.3-80 nM) fully inhibited the neuronal activity of LC cells, with an EC₅₀ value of 15 nM (n = 9). The EP3 receptor antagonist L-798,106 (10 µM) caused a rightward shift (> 8 fold) in the concentration effect curve for sulprostone, but the EP2 receptor antagonist PF04418948 (10 µM) or the EP4 receptor antagonist L-161,982 (10 µM) failed to cause any rightward shift of sulprostone effect. On the other hand, perfusion with the endogenous PGE₂ (0.3 nM-1.28 µM) or the PGE₁ analogous misoprostol (0.3-320 nM) induced a concentration-dependent inhibition of the firing rate of LC cells, with EC₅₀ values being 51 nM and 112 nM respectively. Likewise, only the EP3 antagonist L-798,106 (10 µM) caused a rightward shift (> 8 fold) in the concentration-effect curves for these prostanoid agonists (n = 5). In conclusion, LC neurons are regulated in an inhibitory manner by the prostanoid system likely through the EP3 receptor.