Many membrane-bound neurotransmitter receptors are known to be internalized by exposure to agonist. This agonist-induced receptor internalization is considered to play important roles in receptor-mediated signaling. Here we investigated the internalization of GAR-3, a Caenorhabditis elegans muscarinic acetylcholine receptor, using cultured mammalian cells. When Chinese hamster ovary cells stably expressing GAR-3 were treated with carbachol, GAR-3 was internalized in a dose- and time-dependent manner. Approximately 60% of the cell surface receptor was internalized by exposure to 1 mM carbachol for 1 h. Carbachol-induced GAR-3 internalization was suppressed by treatment with hypertonic sucrose, which blocks the formation of clathrin-coated pits. Overexpression of a dominant-negative dynamin mutant (DynK44A), but not of a dominant-negative beta-arrestin mutant (Arr319-418), substantially inhibited carbachol-induced internalization of GAR-3. Thus, these data suggest that GAR-3 undergoes agonist-induced internalization via a clathrin- and dynamin-dependent but beta-arrestin-independent pathway. Depletion of Ca2+ by simultaneous treatment of the cells with BAPTA/AM (Ca2+ mobilization blocker) and EGTA (Ca2+ influx blocker) almost completely blocked agonist-induced GAR-3 internalization. Moreover, treatment of the cells with the Ca2+ ionophore A23187 led to GAR-3 internalization in the absence of agonist. These results indicate that Ca2+ plays a critical role in GAR-3 internalization. We tested whether the third intracellular (i3) loop of GAR-3 is involved in agonist-stimulated receptor internalization. A GAR-3 deletion mutant lacking a large central portion of the i3 loop exhibited an internalization pattern comparable to that of the wild type, suggesting that the central i3 loop is not required for the internalization of GAR-3.