SummaryCholesterol depletion reversibly abolishes carbachol-evoked Ca 2+ release from inositol (1,4,5)-trisphosphate (IP 3 )-sensitive stores, without affecting the distribution of IP 3 receptors (IP 3 R) or endoplasmic reticulum, IP 3 formation or responses to photolysis of caged IP 3 . Receptors that stimulate cAMP formation do not alone evoke Ca 2+ signals, but they potentiate those evoked by carbachol. We show that these potentiated signals are entirely unaffected by cholesterol depletion and that, within individual cells, different IP 3 -sensitive Ca 2+ stores are released by carbachol alone and by carbachol combined with receptors that stimulate cAMP formation. We suggest that muscarinic acetylcholine receptors in lipid rafts deliver IP 3 at high concentration to associated IP 3 R, stimulating them to release Ca 2+ . Muscarinic receptors outside rafts are less closely associated with IP 3 R and provide insufficient local IP 3 to activate IP 3 R directly. These IP 3 R, probably type 2 IP 3 R within a discrete Ca 2+ store, are activated only when their sensitivity is increased by cAMP. Sensitization of IP 3 R by cAMP extends the effective range of signalling by phospholipase C, allowing muscarinic receptors that are otherwise ineffective to recruit additional IP 3 -sensitive Ca 2+ stores.