In human SH-SY5Y neuroblastoma cells, carbachol stimulates inositol phospholipid breakdown with an EC 50 value of 18 μM. The response is prevented by pirenzepine, with a Hill coefficient of 0.57 and an IC 50 value of 0.15 μM. A Gpp[NH]p-stimulated [ 3H]phosphatidylinositol-4,5-bisphosphate hydrolysing activity was demonstrated in SH-SY5Y membrane preparations. Treatment of the cells for 3 days with retinoic acid (0.1 and 1 μM, in 1% ethanol) caused them to differentiate. The ethanol per se increased the incorporation of tritium into the inositol phospholipids following incubation of the cells with [ 3H]myo-inositol. The inositol phospholipid response to carbachol (1000 μM) was lower following treatment for 3 days with 1 μM retinoic acid than with 0.1 μM retinoic acid. The EC 50 values for carbachol, relative rates of stimulation (with respect to that produced by 1000 μM carbachol) with arecoline, oxotremorine-M and oxotremorine, lack of synergy between carbachol and raised [K +] were the same in undifferentiated and retinoic acid-differentiated cells. It is concluded that (a) more than one muscarinic receptor type is coupled to the inositol phospholipid breakdown response in undifferentiated SH-SY5Y cells, and (b) retinoic acid-induced differentiation of the cells does not affect the properties of the muscarinic receptors coupled to the response, although the magnitude of the response appears to be sensitive to the retinoic acid concentration used to induce the cell differentiation.