Tetanus toxin (100 nM) when preincubated with guinea pig cerebrocortical synaptosomes for 45 min reduces the final extent of the KCl-evoked, Ca(2+)-dependent, glutamate transmitter release to 30% of non-intoxicated controls. Similarly, 100 nM Botulinum neurotoxins, types A and B, preincubated for 90 min inhibit release to 45-60% of non-intoxicated controls. The toxins preferentially attenuate a slow phase of KCl-evoked glutamate release which may be associated with synaptic vesicle mobilization. Tetanus toxin additionally inhibits the release of aspartate, gamma-aminobutyric acid and met-enkephalin from the same preparation. Since amino acids and neuropeptides are released by distinct mechanisms, this indicates that the toxin affects a step common to both exocytotic pathways. When Ba2+ (which does not interact with calmodulin) is substituted for Ca2+, the control KCl-evoked release of each transmitter is unaffected and tetanus toxin is still inhibitory. Taken together these results implicate a calmodulin-independent locus (or loci) of action common to small- and large-dense-core vesicles and associated with vesicle transport.