We previously showed that serotonin (5-HT) produces superoxide anion (O2●) in the intrapulmonary artery (IPA) smooth muscle. Then, O2● passes through the myoendothelial junction (connexin (Cx) 43) to decrease the bioavailability of endothelial NO and strengthen IPA vasoreactivity (Billaud et al., 2009). Here, we further addressed the signalling pathways involved in such process. Male Wistar rats were humanely killed according to national guidelines. IPA were used for immunofluorescence labellings and recording of O2● levels by electron paramagnetic resonance (EPR). Mitochondrial Ca2+ (Ca2+m) was assessed in smooth muscle cells with Rhod-2. Isometric contraction was recorded on IPA rings with an organ bath system. Results were expressed as mean ± S.E.M. Significance was considered when P<0.05 and tested with Mann-Whitney (unpaired samples) and Student t-Test (paired samples). n represents the number of rats for EPR and immunofluorescence, the number of arterial rings for the contractile studies and the number of cells for Ca2+m recordings. 5-HT (100 µM) significantly increased O2● levels in rat IPA (8672.8 ± 752.9 arbitrary units per mg.ml-1 of proteins in response to 5-HT vs 5205 ± 589.7 in basal conditions, n=12-13). Endothelin-1 (ET-1) (0.1 µM) and phenylephrine (Phe) (10 µM) had no effect. Ketanserin (1 µM), a blocker of 5-HT2A receptors significantly decreased 5-HT-induced O2● production whereas blocking the 5-HT1B receptors and the 5-HT transporter (5-HTT) by GR 127935 (1 µM) or citalopram (1 µM) respectively had no effect (n=11-12). EPR recordings showed that removal of extracellular Ca2+ (Ca2+e) decreased 5-HT-induced O2● increase (6310 ± 585.8, p=0.016) whereas depleting intracellular Ca2+ stores with thapsigargin (1 µM) had no effect (n=12-13). Blockers of O2● sources namely apocynin (30 µM) and rotenone (5 µM) for NADPH oxidase (Nox) and the complex I of the mitochondrial respiratory chain (MRC I) respectively, reduced 5-HT-induced O2● increase (n = 10-19). 5-HT enhanced the Ca2+m level with or without Ca2+e (n=30-40). Inhibition of PKCε significantly reduced O2● production to its basal level (n=8-9). Unlike 5-HT1B and ETB receptors, both 5-HT2A and α1D receptors (Phe receptors) colocalised with Cx 43. 5-HT2A receptors and Cx 43 also colocalised with caveolin-1. Unlike the contraction to ET-1, the contraction to 5-HT and Phe was significantly modified after caveolae removal with methyl-β-cyclodextrin (7 mM) (n=12-26). Altogether, 5-HT acts on 5-HT2A receptors and induces a calcium influx responsible for Ca2+m increase and O2● production (via MRC I). This process activates PKCε which in turn may activate O2● production by Nox. Interestingly, O2● production in rat IPA seems to be 5-HT specific and would happen in caveolae localised near to Cx43.