1. Differentiation of SH-SY5Y neuroblastoma cells induces morphological and biochemical changes consistent with a more neuronal phenotype. These cells may therefore provide a model for studying phenomena such as signal transduction in a neuronal context whilst retaining the advantages of a homogenous cell population expressing a well characterized array of G-protein coupled receptors. 2. This study examined the effects of differentiating SH-SY5Y cells on muscarinic- and bradykinin-receptor-mediated phosphoinositide and Ca2+ signalling. Retinoic acid (10 microM, 6 days) along with a lowered serum concentration produced phenotypic changes consistent with differentiation including reduced proliferation and increased neurite outgrowth. 3. Differentiation increased the magnitude and potency of rapid Ins(1,4,5)P3 responses to a full muscarinic receptor agonist. Bradykinin receptor-mediated Ins(1,4,5)P3 signalling was also potentiated following differentiation. Determination of agonist-evoked accumulation of [3H]-inositol phosphates under lithium-block demonstrated these changes reflected enhanced phospholipase C activity which is consistent with observed increases in the expression of muscarinic and bradykinin receptors. 4. Despite the marked alterations in Ins(1,4,5)P3 signalling following differentiation, elevations of intracellular [Ca2+] were totally unaltered. Thus, in SH-SY5Y cells, the relationship between the elevations of Ins(1,4,5)P3 and intracellular [Ca2+] is agonist dependent and affected by the state of differentiation. This demonstrates that mechanisms other than the measured increase in Ins(1,4,5)P3 regulate the elevation of intracellular [Ca2+].