Truncation of the C-terminal domain of the synaptosomal associated protein of 25 kDa (SNAP-25) by botulinum neurotoxin A (BoNT A) has been shown to block neuroendocrine cell secretion. It is unclear, however, if toxin mechanism involved the affection of a single or more events of the exocytotic cascade. BoNT A induced changes in both the degree of inhibition and the kinetics of catecholamine secretion from populations of cultured bovine chromaffin cells. Ca2+-dependent secretion from digitonin-permeabilized cells showed partial inhibition associated with the alteration of a slow secretory phase at different toxin concentrations. In contrast, in intact cells stimulated by depolarization, cell treatment with low concentrations (1 nm) of the toxin affected the late phase of secretion, whereas 100 nm BoNT A-poisoned cells showed an alteration even of fast components. The high degree of inhibition associated with fast secretory component alteration was dependent on Ca2+ entry through the Ca2+ channels, as it was absent from cells permeated with the A23187 Ca2+ ionophore. Vesicle pools implicated in the effect of BoNT A on the secretory response from single cells were identified using amperometry. These studies supported the macroscopic view by showing that secretion from BoNT A-treated permeabilized cells presented specific inhibition of late vesicle fusions. Intact cells showed alterations in the late vesicle pool (t = 39 s) recruited during prolonged or repetitive KCl depolarizations using 1 nm BoNT A-treated cells as well as in an intermediate kinetic pool (t = 18 s) at higher toxin concentrations (100 nm). The faster resolved component (t = 8 s) or the membrane fusion event itself were not affected. Our results demonstrate that removal of the last nine C-terminal amino acids of SNAP-25 by BoNT A has a specific effect on two different and distal secretory stages in chromaffin cells.