At the frog neuromuscular junction, neurotransmitter release sites are regularly spaced at 1 micron intervals along the nerve terminal, directly facing postsynaptic folds which contain a high density of acetylcholine receptors. Immunostaining and laser confocal scanning microscopy were used to compare the distribution of presynaptic proteins implicated in exocytosis with that of fluorescent alpha-bungarotoxin. Syntaxin, synaptosome-associated 25 kDa protein and calcium channels were located predominantly at release sites. Synaptobrevin (vesicle-associated membrane protein) was distributed in the cytoplasm of the nerve terminal, presumably in the packets of microvesicles associated with each active zone. N-Ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (alpha beta SNAP) displayed a diffuse distribution throughout the terminal cytoplasm and also colocalized in distinct concentrated zones adjacent to the presynaptic membrane.