Cyclic AMP (cAMP) synthesis via adenylate cyclase (AC) plays a crucial role in the vasodilatory effects of β-adrenoceptor stimulation in the vasculature. Stimulation of cAMP-dependent protein kinase (PKA) activates a variety of K+ channels present in vascular smooth muscle cells (SMCs), leading to hyperpolarisation and vasodilatation 1. Despite the central importance of the AC enzyme in this pathway, it is not known which isoenzyme(s) mediates cAMP synthesis in response to vasodilators in vascular smooth muscle, where several distinct isoenzymes have been detected 2-4. Using real-time RT-PCR, we have identified that in SMCs acutely isolated from rat aorta and mesenteric artery, the predominant AC isoforms present (at the mRNA level) are types 3, 5, 6 and 7. A similar profile was observed in cultured rat aortic SMCs. In order selectively to manipulate endogenous AC isoenzymes, siRNA sequences targeting each of these four AC isoforms (3, 5, 6 and 7) or a scrambled control siRNA were transfected into cultured rat aortic SMCs. AC isoenzyme expression was then assessed (relative to control, 48 h post-transfection) by RT-PCR. For each of the four AC isoenzymes, siRNA sequences that reduced expression of their target isoenzyme by ≥75 % (with no significant effects on the expression level of non-targeted AC isoenzymes) were identified. For AC3, reduced expression level was also confirmed by western blotting with an AC3-specific antibody. cAMP levels were assessed in siRNA (control or AC-targeting)-transfected aortic SMCs in response to the β-adrenoceptor agonist isoprenaline (Iso, 1 µM) in the presence of the non-selective phosphodiesterase 3-isobutyl-1-methylxanthine (IBMX, 300 µM). Iso-mediated cAMP responses were not significantly altered by depletion of AC7 (129 ± 8% of control; n = 4). In contrast, in SMCs transfected with siRNA targeting ACs 3, 5 and 6, cAMP responses to Iso in the presence of IBMX were reduced by >75%, while responses to Iso alone were reduced by 88%. We also investigated the role of each individual AC isoenzyme in β-adrenoceptor-mediated cAMP generation. Depletion of AC6 had the greatest effect on Iso-stimulated cAMP accumulation (reducing it to 41 ± 10% of control; n=5, p<0.01, 2-way ANOVA), while AC3 knockdown also significantly decreased the response, to 70 ± 3% of control (n=6, p<0.01, 2-way ANOVA). Depletion of AC5 slightly reduced cAMP accumulation in response to Iso (to 79 ± 14%; n=4), but this did not achieve statistical significance. In conclusion, we have shown that AC6 plays a predominant role in β-adrenoceptor-mediated cAMP signalling in vascular SMCs, while AC3 and perhaps AC5 play lesser roles in mediating the cAMP response to this vasodilator.