Internalization and phosphorylation of G protein-coupled receptors (GPCR) are considered two important regulatory events of receptor signal transduction. In Fischer rat thyroid (FRT) epithelial cells, we have shown that muscarinic acetylcholine receptor (mAChR) stimulation induces intracellular Ca 2+ mobilization via Ca 2+ store release, capacitative Ca 2+ entry and voltage-dependent Ca 2+ channels activation. In the present study, the role of mAChR internalization and phosphorylation on receptor signalling pathway was examined by means of intracellular Ca 2+ measurement in these cells. Exposure of FRT cells to carbachol (Cch), a mAChR agonist, resulted in a desensitization of receptor-mediated intracellular Ca 2+ mobilization and induced the internalization of constitutively expressed mAChR in this cell type. Treatment of FRT cells with hypertonic sucrose, which markedly reduced agonist–receptor complex internalization, or phenylarsine oxide (PAO) diminished the Cch-induced intracellular Ca 2+ response. Moreover, pretreatment of cells with phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC), completely abolished Cch-evoked Ca 2+ mobilization, whereas it was significantly increased by the preincubation of cells with GF109203X, a selective inhibitor of PKC. We also found a marked decrease on Cch-stimulated Ca 2+ mobilization in pretreated FRT cells with forskolin, an activator of protein kinase A (PKA), but the preincubation of cells with genistein, an inhibitor of protein tyrosine kinases, had no effect on Ca 2+ mobilization induced by Cch. These findings seem to indicate that mAChR in FRT cells exhibit a desensitization, which may be mediated, at least in part, through activation of second messenger-dependent protein kinases and that receptor internalization could be necessary for signalling.