The present study examines the paralytic action of botulinum neurotoxins at their natural target, the neuromuscular junction. We asked whether syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicle-associated membrane protein (VAMP/synaptobrevin), the proteins proteolysed by botulinum, are susceptible to cleavage in frog nerve terminals, and whether they form complexes in vivo. In control terminals, the three SNAREs were distributed in broad bands at 1 micrometer intervals, at sites consistent with presynaptic Ca2+ channels. Within 3 h, botulinum A, C, D and E (BoNT/A/C/D/E) blocked nerve-evoked muscle contractions but their effects on substrate immunoreactivity varied. The effect of BoNT/A on either C-terminus or N-terminus immunoreactivity of SNAP-25 was undetectable after 3-h incubation, although C-terminus immunoreactivity was reduced after 24 h; N-terminus immunoreactivity was not affected even after 36 h. BoNT/E reduced C-terminus immunoreactivity of SNAP-25 1.5 h after toxin application when transmitter release was blocked, but required 24 h to reduce N-terminus immunoreactivity. BoNT/C reduced syntaxin immunoreactivity after 24-h incubation but did not affect SNAP-25. BoNT/D reduced VAMP immunoreactivity at 3 h while it increased SNAP-25 C-terminal staining fourfold. BoNT/A and BoNT/C applied together for 24 h reduced syntaxin immunoreactivity and that of both C- and N-terminus of SNAP-25, indicating that retention of SNAP-25 N-terminus after cleavage by BoNT/A depended on intact syntaxin. Therefore, we infer that SNAP-25 interacts with VAMP and with syntaxin in vivo. Neurotoxin action abolished only 40-60% of SNAP-25, VAMP or syntaxin immunoreactivity suggesting that distinct pools of these proteins, not immediately involved in triggered exocytosis, are resistant to proteolysis.