The recovery of the muscarinic cholinergic receptor (mAChR) from its down-regulation by long-term exposure to ACh was investigated. This was done to obtain information about regulation of the mAChR. Exposure of guinea-pig vas deferens to 30 microM ACh for 24 h decreased the amount of mAChR to 30% of the initial level, as measured with L-[3H]quinuclidinyl benzilate (QNB). The amount of mAChR was restored to 190% of its prewithdrawal level within 48 h of removal of ACh, without change in the KD value for L-[3H]QNB. This restoration was entirely dependent on protein synthesis. The half-life of the receptor was calculated to be 69 h. The recovery of mAChR was blocked by treatment with antimicrotubular agents, carboxylic ionophores, or 5 mM EGTA, which affect membrane protein synthesis. However, cytochalasin B and cyclic nucleotide derivatives had no effect. These data indicate that the recovery of mAChR was due to new synthesis of mAChR. The findings suggested that microtubules and the Golgi apparatus were involved in the biosynthesis of mAChR and that extracellular Ca2+ was necessary for the synthesis. Unlike the case with nicotinic AChR, the synthesis did not seem to be increased by cAMP or high extracellular Ca2+.