Synaptic transmission between baroreceptor afferents and the nucleus tractus solitarius (NTS) is known to exhibit frequency-dependent depression. Reductions in neurotransmitter release and alterations in mechanisms regulating synaptic transmission are hypothesized to be involved in the activity-dependent depression observed in baroreceptor afferent neurons. The present study utilized cultured aortic baroreceptor neurons and the fluorescent dyes FM1-43 and FM2-10 to characterize the process of endocytosis or vesicle retrieval and its dependence on 1) frequency of neuronal activation, 2) metabotropic glutamate receptor (mGluR) activation, and 3) calcium concentrations inside and outside the cell. Endocytosis per spike, measured in fluorescence units after a 10-s stimulus applied at frequencies of 0.5 (53 ± 4), 1.0 (23 ± 1), and 10.0 Hz (2.7 ± 0.2), was significantly depressed at higher frequencies. Blockade of group III mGluRs with (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG) facilitated endocytosis at all frequencies, suggesting that this receptor subtype may be involved in the inhibition of endocytosis. Manipulating the extracellular and intracellular calcium concentrations subsequent to exocytosis had no effect on endocytosis. These results suggest that frequency-dependent depression of endocytosis observed in vitro could contribute to the frequency-dependent depression of baroreceptor afferent neurotransmission and that group III mGluRs inhibit endocytosis.