Adenine and uridine nucleotides evoke Ca2+ signals via four subtypes of P2Y receptor in cultured aortic smooth muscle cells, but the mechanisms underlying the different patterns of these Ca2+ signals are unresolved. Cytosolic Ca2+ signals were recorded from single cells and populations of cultured rat aortic smooth muscle cells, loaded with a fluorescent Ca2+ indicator and stimulated with agonists that allow subtype-selective activation of P2Y1, P2Y2, P2Y4, or P2Y6 receptors. Activation of P2Y1, P2Y2, and P2Y6 receptors caused homologous desensitisation, while activation of P2Y2 receptors also caused heterologous desensitisation of the other subtypes. The Ca2+ signals evoked by each P2Y receptor subtype required activation of phospholipase C and release of Ca2+ from intracellular stores via inositol 1,4,5-trisphosphate (IP3) receptors, but they were unaffected by inhibition of ryanodine or nicotinic acid adenine dinucleotide phosphate (NAADP) receptors. Sustained Ca2+ signals were independent of the Na+/Ca2+ exchanger and were probably mediated by store-operated Ca2+ entry. Analyses of single cells established that most cells express P2Y2 receptors and at least two other P2Y receptor subtypes. We conclude that four P2Y receptor subtypes evoke Ca2+ signals in cultured aortic smooth muscle cells using the same intracellular (IP3 receptors) and Ca2+ entry pathways (store-operated Ca2+ entry). Different rates of homologous desensitisation and different levels of receptor expression account for the different patterns of Ca2+ signal evoked by each P2Y receptor subtype. © 2012 Springer Science+Business Media B.V.