G protein coupled receptors (GPCR) play important roles in controlling neurotransmitter and hormone release. Inhibition of voltage-gated Ca²⁺ channels (Ca²⁺-channels) by G protein subunits (G) is one prominent mechanism but there is evidence for additional effects distinct from those on calcium entry. However, relatively few studies have investigated the Ca²⁺-channel independent effects of G on transmitter release so the impact of this mechanism remains unclear. We used carbon fiber amperometry to analyze catecholamine release from individual vesicles in bovine adrenal chromaffin cells, a widely used neurosecretory model. To bypass the effects of G on Ca²⁺ entry we stimulated secretion using ionomycin (a Ca²⁺-ionophore) or direct intracellular application of Ca²⁺ through a patch-pipette. Activation of endogenous GPCR or transient transfection with exogenous G significantly reduced the number of amperometric spikes (the number of vesicular fusion events). The charge ("quantal size") and amplitude of the amperometric spikes were also significantly reduced by GPCR / G. We conclude that, independent from effects on calcium entry, G can regulate both the number of vesicles that undergo exocytosis and the amount of catecholamine released per fusion event. We discuss possible mechanisms by which G might exert these novel effects including interaction with the SNARE complex.