Trachynilysin, a neurotoxin isolated from the venom of the stonefish (Synanceia trachynis, Scorpaenidae), produced a marked increase in the frequency of spontaneous miniature endplate potentials (MEPPs) at Torpedo neuromuscular junctions. The periods of high frequency MEPP discharges were of variable duration and were followed by periods of rest. In addition, trachynilysin increased as a function of time the proportion of larger than normal MEPPs, the so-called “giant” MEPPs. Trachynilysin did not affect the junctions when applied in Ca2+-free medium supplemented with EGTA, but the subsequent addition of Ca2+ caused a rapid increase in MEPP frequency, even when the toxin was washed out of the Ca2+-free medium. Thus, trachynilysin binding to nerve terminals is not dependent on external Ca2+, but the cation is required for trachynilysin-elicited quantal transmitter release. The effect of trachynilysin on MEPP frequency was unaffected by the Ca2+ channel blockers w-conotoxin GVIA, w-agatoxin IVA and Gd3+, which indicates that the toxin’s action involves Ca2+ entry via a pathway independent of voltage-sensitive Ca2+ channels. Pre-treatment of the junctions with concanavalin-A prevented the trachynilysin-induced enhancement of quantal transmitter release, which suggests that the toxin interacts with or binds to a glycoprotein on the surface of motor nerve terminals.