We have used the proteolytic properties of botulinum and tetanus neurotoxins (BoNT, TeNT) to cleave three proteins of the membrane fusion machinery, SNAP-25, VAMP/synaptobrevin, and syntaxin, in developing and differentiated rat central neurons in vitro. Then, we have studied the capacity of neurons to extend neurites, make synapses, and release neurotransmitters. All the toxins showed the expected specificity with the exception that BoNT/C cleaved SNAP-25 in addition to syntaxin and induced rapid neuronal death. In developing neurons, cleavage of SNAP-25 with BoNT/A inhibited axonal growth and prevented synapse formation. In contrast, cleavage of VAMP with TeNT or BoNT/B had no effects on neurite extension and synaptogenesis. All the toxins tested inhibited transmitter release in differentiated neurons, and cleavage of VAMP resulted in the strongest inhibition. These data indicate that SNAP-25 is involved in vesicle fusion for membrane expansion and transmitter release, whereas VAMP is selectively involved in transmitter release. In addition, our results support the hypothesis that synaptic activity is not essential for synapse formation in vitro.