Mice over-expressing the serotonin transporter (5-HTT+) are pre-disposed to hypoxia-induced pulmonary hypertension.¹ Pulmonary arteries removed from 5-HTT+ mice, however, exhibit reduced sensitivity to 5-HT-induced contraction when compared to wild-type (WT). We investigated the possible mechanisms behind this by examining the influences of membrane potential and Ca²⁺ entry in intrapulmonary arteries (IPAs) from WT and 5-HTT+ mice. Mice (C57BL/6, 5-6 month, 25-40g) were euthanised (sodium pentobarbitone 2g/kg) and IPAs dissected out and mounted on wire myographs (Krebs; 37°C). Cumulative concentration-response curves were determined for 5-HT (10^-9M-10^-4M) in the presence and absence of nifedipine (inhibits voltage-operated Ca²⁺ channels), NiCl₂ (inhibits store-operated Ca²⁺ entry, SOCE), 2-APB (inhibits IP3-induced Ca²⁺ release and SOCE) and at different extracellular K⁺ concentrations (5, 20 and 50mM). Responses to 10μM 5-HT were also examined in Ca²⁺-free solution. Contractile responses are expressed as % response to 50mM KCl. Statistical analysis used Students t-test, p<0.05 considered significant. 5-HT was more potent in WT compared to 5-HTT+ (pEC50 = 6.9±0.08, n=12 and 5.3±0.1, n=6, respectively, P<0.001). Removing extracellular Ca²⁺ abolished the contraction induced by submaximal 5-HT (10µM) in both WT (2.4±3.7% cf. control 60±9% n=6, P<0.001) and 5-HTT+ (8±2% cf. control 70±7% n=5, P<0.001). Nifedipine (5μM) partially inhibited 5-HT-induced contraction in WT and 5-HTT+ mice, significantly reducing the maximum response (Emax): WT: 98±7% cf. 64±5%, P<0.01; 5-HTT+: 86±5% cf. 59±2%, P<0.001. In nifedipine pEC50 values were 5.8±0.2, n=7 (WT) and 5.29±0.06, n=6 (5-HTT+), P<0.05. NiCl₂ (50μM) reduced pEC50 values in both preparations: WT pEC50= 6.75±0.06 n=6, cf. 7.0±0.1 n=6 P<0.001; 5-HTT+ pEC50= 5.94±0.04 n=8, cf. 5.65±0.05 n=8, P<0.001. 2-APB (75μM) reduced the Emax of 5-HT-induced contraction in WT & 5-HTT+ vessels: WT Emax= 88.7±0.2% n=6, cf. 46.6±0.1% n=7, P<0.001; 5-HTT+ Emax= 112.5±0.2%, n=5 cf. 61.2±0.4%, n=6, P<0.001. 5-HT induced contractions remained significantly different in WT and 5-HTT+ at different K⁺ concentrations. In 5mM K⁺ pEC50= 6.97±0.08 n=7 (WT) cf. 5.74±00.08 n=6 (5-HTT+), P<0.001. Potency increased in 20mM K⁺+ to pEC50=7.6±0.1, n=6 (WT) cf. 5.98±0.07 n=6 (5-HTT+), P<0.001, but fell in 50mM K⁺ to pEC50=7.2±0.2 n=4 (WT) cf. 5.8±0.2 n=6 (5-HTT+), P<0.001. The results suggest that 5-HT-induced contraction requires Ca²⁺ influx and involves both voltage-dependent Ca²⁺ channels and SOCE. The difference in 5-HT potency between WT and 5-HTT+ is not related to membrane potential as it was not restored by changing extracellular K⁺. No difference in Ca²⁺ transport was found between WT and 5-HTT+ IPAs to explain the difference in sensitivity to 5-HT.