Neuropeptide Y, alpha 2-adrenoceptors, thrombin and certain muscarinic acetylcholine receptors can couple to elevations of intracellular free Ca2+ concentrations via Gi-proteins. We have studied the effects of inhibitors of microtubules (colchicine, nocodazole, vinblastine) and microfilaments (cytochalasin B, cytochalasin D) on these effects in human erythroleukemia (HEL) cells. Both types of inhibitors reduced neuropeptide Y-, adrenaline- (via alpha 2A-adrenoceptors) and thrombin-stimulated Ca2+ elevations while the inactive analog beta-lumicolchicine was without inhibitory effects. Similarly, in SK-N-MC cells vinblastine inhibited neuropeptide Y and carbachol (via muscarinic receptors) stimulated Ca2+ elevations. In HEL cells the inhibitory effects of the microfilament inhibitor cytochalasin D and the microtubule inhibitor colchicine were not additive. Colchicine, nocodazole or cytochalasin D did not affect the binding of the agonist neuropeptide Y. On the other hand, neuropeptide Y and thrombin significantly stimulated GTP gamma S binding in the absence but not in the presence of colchicine, vinblastine or cytochalasin B. This was not due to sequestration of G-protein alpha-subunits, since nocodazole did not affect the distribution of immunodetectable Gi alpha 1/2 or Gi alpha 3 between membrane and cytosolic fractions. We conclude that disruption of microfilaments or microtubules impairs Ca2+ elevations by neuropeptide Y and other Gi-coupled receptors by inhibiting receptor/Gi-protein interaction; this does not involve impairment of agonist binding to the receptor or redistribution of Gi-protein alpha-subunits.