The effects of activation of muscarinic receptors on chromaffin cells and splanchnic nerve terminals were studied in a rat adrenal slice preparation. In chromaffin cells, muscarine induced a transient hyperpolarization followed by a depolarization associated with cell spiking. The hyperpolarization was blocked by charybdotoxin (1 μm) and tetraethylammonium chloride (TEA, 1 mm), but was not affected by 200 μm Cd2+ or removal of external Ca2+, consistent with activation of BK channels. This would follow internal Ca2+ mobilization, as shown by Ca2+ imaging with fura-2 on isolated chromaffin cells in culture. Under voltage-clamp, outward BK currents were insensitive to MT3 toxin, a specific muscarinic m4 receptor antagonist. In contrast, muscarine-induced depolarization was due to a m4 receptor-mediated inward current blocked by MT3 toxin. This current was permeable to cations and was associated with Ca2+ entry and subsequently, Ca2+-induced Ca2+ release. Finally, both muscarine (25 μm) and oxotremorine (10 μm) decreased the amplitude and frequency of KCl-evoked excitatory postsynaptic currents, without affecting quantal size, consistent with a presynaptic inhibitory effect. Taken together, our data suggest that activation of m4 and probably m3 muscarinic receptors results in a strong, long-lasting excitation of chromaffin cells, as well as an uncoupling of synaptic inputs onto these cells.