Our previous observation that low concentrations of stonefish (Synanceia trachynis) venom elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals prompted our present study to purify the quantal transmitter-releasing toxin present in the venom and to characterize the toxin's ability to alter the ultrastructure and immunoreactivity of frog motor nerve terminals. Fractionation of S. trachynis venom by sequential anion exchange fast protein-liquid chromatography (FPLC) and size-exclusion FPLC yielded a highly purified preparation of a membrane-perturbing (haemolytic) protein toxin, named trachynilysin. Trachynilysin (2–20 μg/ml) significantly increased spontaneous quantal acetylcholine release from motor endings, as detected by recording miniature endplate potentials from isolated frog cutaneous pectoris neuromuscular preparations. Ultrastructural analysis of nerve terminals in which quantal acetylcholine release was stimulated to exhaustion by 3 h exposure to trachynilysin revealed swelling of nerve terminals and marked depletion of small clear synaptic vesicles. However, trachynilysin did not induce a parallel depletion of large dense-core vesicles. Large dense-core vesicles contained calcitonin gene-related peptide (CGRP), as revealed by colloidal gold immunostaining, and trachynilysin-treated nerve endings exhibited CGRP-like immunofluorescence similar to that of untreated terminals. Our results indicate that the ability of stonefish venom to elicit spontaneous quantal acetylcholine release from vertebrate motor nerve terminals is a function of trachynilysin, which selectively stimulates the release of small clear synaptic vesicles and impairs the recycling of small clear synaptic vesicles but does not affect the release of large dense-core vesicles. Trachynilysin may be a valuable tool for use in other secretory terminals to discriminate between neurotransmitter and neuropeptide release.