Susceptibilities of Mg.ATP-independent and Mg.ATP-requiring components of catecholamine secretion from digitonin-permeabilised chromaffin cells to inhibition by Clostridial botulinum type A and tetanus toxins were investigated. These toxins are Zn(2+)-dependent proteases which specifically cleave the 25-kDa synaptosomal-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) II, respectively. When applied to permeabilised chromaffin cells they rapidly inhibited secretion in the presence of Mg.ATP but the catecholamine released in the absence of Mg.ATP, thought to represent fusion of primed granules, was not perturbed. The toxins can exert their effects per se in the absence of the nucleotide complex; therefore, Mg.ATP-requiring steps of secretion are implicated as roles for their targets. Primed release was lost rapidly after permeabilisation of the cells but could be maintained by including Mg.ATP during the incubation before stimulating release with Ca2+. This ability of Mg.ATP to maintain primed release was only partially inhibited by botulinum neurotoxin A whereas it was abolished by tetanus toxin, consistent with the distinct substrates for these toxins. This study reveals a component of release within which these proteins are either resistant to cleavage by these toxins or in such a position that degradation can no longer prevent granule fusion. Differences in the steps of release at which these toxins can affect inhibition are also revealed.