Vascular smooth muscle cells form most of the walls of blood vessels and their contractile ability determines blood flow and blood pressure. During vascular injury (eg. atherosclerosis), they can also proliferate and exhibit the synthetic phenotype. Elevations of intracellular Ca²⁺ initiate contraction, while during vascular injury they may promote cell growth. cAMP has opposing effects on smooth muscle cell activity and interactions between Ca²⁺ and cAMP are clearly important. Here, we have characterised Ca²⁺ signals evoked by the purinergic receptor agonist, ATP in cultured rat aortic smooth muscle cells (RASMCs). We have investigated whether increases in cytosolic Ca²⁺ concentration affect adenylyl cyclase (AC) activity. For Ca²⁺ measurements, cells were loaded with the Ca²⁺ indicator fluo-4 and fluorescent measurements were made in a 96-well population based flexstation assay. All results are expressed as means + S.E. ATP mobilized Ca²⁺ in a concentration-dependent manner (EC₅₀ = 3.5 + 1.1 µM, n = 3) and the peak response was unchanged in the absence of extracellular Ca²⁺. Involvement of P2Y purinergic receptors was implicated because the response to ATP was completely abolished by the phospholipase C inhibitor, U73122 (IC₅₀ = 1.7 + 0.27µM, n = 3) and there was no response to the P2X receptor agonist, α,β-methyl ATP. cAMP measurements were made using radioimmuno assay and all AC stimulations were made in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). ATP (100µM) inhibited forskolin-stimulated AC by 24 + 1.8% (n = 4, p = 0.001, student’s t-test). However, this inhibition persisted in the absence of extracellular Ca²⁺ (31 + 1.02%, n = 3, p = 0.0022) and after intracellular Ca²⁺ stores were emptied using thapsigargin (1µM) (29 + 2.04%, n = 3, p = 0.005). This effect was abrogated after treatment of cells with pertussis toxin (n = 3). To summarise, ATP mobilized intracellular Ca²⁺ via P2Y receptors and inhibited forskolin-stimulated AC activity in RASMCs. The inhibition of AC activity was independent of increases in cytosolic [Ca²⁺] and is likely to be mediated by Gi G-protein.