The ability of receptors coupled to phosphoinositide turnover to evoke accumulation of inositol 1,4,5-trisphosphate (InsP3) over extended incubation periods, and consequently to affect the level of InsP3 receptor expression, was studied in cultured cerebellar granule cells. The cholinergic agonist carbachol (CCh; 1 mM) evoked a biphasic accumulation of InsP3, a rapid three- to fourfold peak increase over control levels at approximately 10 s, decreasing within 1 min to a long-lasting plateau elevation. Using an antibody against the type I InsP3 receptor, it was demonstrated that > 50% down-regulation of type I InsP3 receptor expression in cerebellar granule cells occurred within 1 h of incubation with 1 mM CCh. Over 24 h, 1 mM CCh caused an approximately 85% decrease in type I InsP3 receptor levels, and significant decreases in immunoreactivity were evident at much lower concentrations of CCh. Direct assessment of total InsP3 receptor expression using a radioligand binding method also detected down-regulation, but to an apparently lesser extent. 1-Aminocyclopentane-1S,3R-dicarboxylic acid (200 microM), an agonist of metabotropic glutamate receptors, evoked a marked decrease in type I InsP3 receptors after 24 h of incubation. These findings demonstrate that a functional consequence of maintained InsP3 production in cerebellar granule cells is the down-regulation of InsP3 receptor expression and that this down-regulation may be a common mechanism of action of phosphoinositide-linked receptors during prolonged stimulation.